Skeletal muscle regeneration is a complex interplay between various cell types including invading macrophages. Their recruitment to damaged tissues upon acute sterile injuries is necessary for necrotic debris clearance and for coordination of tissue regeneration. This highly dynamic process is characterized by an in‐situ transition of infiltrating monocytes from an inflammatory (Ly6Chigh) to a repair (Ly6Clow) macrophage phenotype. The importance of the macrophage phenotypic shift and the cross‐talk of the local muscle tissue with the infiltrating macrophages during tissue regeneration upon injury are not fully understood and their study lacks adequate methodology. Here, using an acute sterile skeletal muscle injury model combined with irradiation, bone marrow transplantation and in vivo imaging we show that preserved muscle integrity and cell composition prior to the injury is necessary for repair macrophage phenotypic transition and subsequently for proper and complete tissue regeneration. Importantly, by using a model of in vivo ablation of PAX7 positive cells, we show that this radiosensitive skeletal muscle progenitor pool contributes to macrophage phenotypic transition following acute sterile muscle injury. In addition, local muscle tissue radioprotection by lead shielding during irradiation preserves normal macrophage transition dynamics and subsequently muscle tissue regeneration. Taken together, our data suggest the existence of a more extensive and reciprocal cross‐talk between muscle tissue compartments, including satellite cells, and infiltrating myeloid cells upon tissue damage. These interactions are shaping the macrophages in‐situ phenotypic shift, which is indispensable for normal muscle tissue repair dynamics.
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Small conductance Ca2+‐activated K+ channels (SK) play an important role in regulating MNCs activity and hormone release from the posterior pituitary. Moreover, an enhanced SK activity contributes to MNCs’ adaptive responses to a physiological challenge, such as lactation. Still, whether changes in SK function/expression contribute to exacerbated MNCs activity during diseases like heart failure (HF), remains unknown. Here, we used a combination of patch‐clamp electrophysiology, confocal Ca2+ imaging and molecular biology in a rat model of ischemic HF. We found that the input‐output function of MNCs was enhanced in HF compared to sham rats. Moreover, while the SK blocker apamin (200 nm) strengthened the input‐output function in sham rats, it failed to have an effect in HF rats. The magnitude of the AHP following a train of spikes, and the underlying apamin‐sensitive IAHP were blunted in MNCs from HF rats. However, spike‐induced increases in intracellular Ca2+ were not affected in MNCs of HF rats. Real‐time PCR measurements of SK channel subunits mRNA in SON punches revealed a diminished expression of SK2/SK3 subunits in HF, compared to sham rats. Together, our studies demonstrate that MNCs from HF rats exhibit increased membrane excitability and an enhanced input‐output function, and that a reduction in SK channel‐mediated, apamin‐sensitive AHP is a critical contributing mechanism. Moreover, our results suggest that the reduced AHP is related to a down‐regulation of SK2/SK3 channel subunit expression, but not due to a blunted activity‐dependent intracellular Ca2+ increase following a burst of action potentials.
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All three epithelial Na+ channel (ENaC) subunits (α, β, and γ) were located in vasopressin (VP) magnocellular neurons in the hypothalamic supraoptic (SON) and paraventricular nuclei. Our previous study demonstrated that ENaC mediates a Na+ leak current that affects the steady state membrane potential in VP neurons. In the present study, we evaluated the effect of dietary salt intake on ENaC regulation and activity in VP neurons. High dietary salt intake for 7 days caused an increase in expression of β‐ and γENaC subunits in the SON and the translocation of αENaC immunoreactivity towards the plasma membrane. Patch‐clamp experiments on hypothalamic slices showed that the mean amplitude of the putative ENaC currents was significantly greater in VP neurons from animals that were fed a high‐salt diet compared with controls. The enhanced ENaC current contributed to the more depolarized basal membrane potential observed in VP neurons in the high‐salt diet group. These findings indicate that high dietary NaCl intake enhances the expression and activity of ENaC which augments synaptic drive by depolarizing the basal membrane potential close to the action potential threshold during hormonal demand. However, ENaCs appear to have only a minor role in the regulation of the firing activity of VP neurons in the absence of synaptic inputs as neither the mean intraburst frequency, burst duration, nor interspike interval variability of phasic bursting activity was affected. Moreover, ENaC activity did not affect the initiation, sustention, or termination of the phasic bursting generated in an intrinsic manner without synaptic inputs.
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The dorsal part of the lateral geniculate nucleus (dLGN) is the main thalamic site for state‐dependent transmission of visual information. Non‐retinal inputs from the ascending arousal system and inhibition provided by γ‐aminobutyric acid (GABA)ergic local circuit interneurons (INs) control neuronal activity within the dLGN. In particular, acetylcholine (ACh) depolarizes thalamocortical relay (TC) neurons by inhibiting two‐pore domain potassium (K2P) channels. Conversely, ACh also hyperpolarizes INs via an as‐yet‐unknown mechanism. By using whole cell patch‐clamp recordings in brain slices and appropriate pharmacological tools we here report that stimulation of type 2 muscarinic ACh receptors (M2AChRs) induces IN hyperpolarization by recruiting the G beta‐gamma complex (Gβγ), class‐1A phosphatidylinositol‐4,5‐bisphosphate 3‐kinase (PI3K), and cellular and sarcoma (c‐Src) tyrosine kinase (TK), leading to activation of two‐pore domain weakly inwardly rectifying K+ channel (TWIK)‐related acid‐sensitive K+ (TASK)‐1 channels. The latter was confirmed by the use of TASK‐1 deficient mice. Furthermore inhibition of phospholipase Cβ (PLCβ) as well as an increase in the intracellular level of phosphatidylinositol‐3,4,5‐trisphosphate (PIP3) facilitated the muscarinic effect. Our results have uncovered a previously unknown role of c‐Src TK in regulating IN function in the brain and identified a novel mechanism by which TASK‐1 channels are activated in neurons.
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Ionotropic glutamate receptors (iGluRs) are tetrameric proteins with between 4 and 12 consensus sites for N‐glycosylation on each subunit, which potentially allows for a high degree of structural diversity conferred by this post‐translational modification. N‐glycosylation is required for proper folding of iGluRs in mammalian cells, but the impact of oligosaccharides on the function of successfully folded receptors is less clear. Glycan moieties are large, polar, occasionally charged, and mediate many protein‐protein interactions throughout the nervous system. Additionally, they are attached at sites along iGluR subunits that position them for involvement in the structural changes underlying gating. We show here that altering glycan content on kainate receptors (KARs) changes the functional properties of the receptors in a manner dependent on the identity of both the modified sugars and the subunit composition of the receptor to which they are attached. We also report that native KARs carry the complex capping oligosaccharide HNK‐1. Glycosylation patterns likely differ between cell types, across development, or with pathologies, and thus our findings reveal a potential mechanism for context‐specific fine‐tuning of KAR function through diversity in glycan structure.
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Ivermectin (IVM) is a widely used antiparasitic drug in humans and pets which activates glutamate‐gated Cl− channel in parasites. It is also known that IVM binds to the transmembrane domains (TMs) of several ligand‐gated channels, such as Cys‐loop receptors and P2X receptors. In this study, we found that the G‐protein‐gated inwardly rectifying K+ (GIRK) channel is activated by IVM directly. By electrophysiological recordings in Xenopus oocytes, we observed that IVM activates GIRK channel in a phosphatidylinositol‐4,5‐biphosphate (PIP2)‐dependent manner, and that the IVM‐mediated GIRK activation is independent of Gβγ. We found that IVM activates GIRK2 more efficiently than GIRK4. In cultured hippocampal neurons, we also observed that IVM activates native GIRK current. By chimeric and mutagenesis analyses, we identified a unique amino acid residue of GIRK2 among GIRK family, Ile82, located in the slide helix between the TM1 and the N‐terminal cytoplasmic tail domain (CTD), which is critical for the activation. The results demonstrate that the TM‐CTD interface in GIRK channel, rather than the TMs, governs IVM‐mediated activation. These findings provide us with novel insights on the action mode of IVM in ion channels, and information toward identification of new pharmacophores which activate GIRK channel.
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Acute O2 sensing is a fundamental property of cells in the peripheral chemoreceptors, e.g. glomus cells in the carotid body (CB) and chromaffin cells in the adrenal medulla (AM), and is necessary for adaptation to hypoxia. These cells contain O2‐sensitive ion channels, which mediate membrane depolarization and transmitter release upon exposure to hypoxia. However, the mechanisms underlying the detection of changes in O2 tension by cells are still poorly understood. Recently, we suggested that CB glomus cells have specific metabolic features that favour the accumulation of reduced quinone and the production of mitochondrial NADH and reactive oxygen species during hypoxia. These signals alter membrane ion channel activity. To investigate the metabolic profile characteristic of acute O2‐sensing cells, we used adult mice to compare the transcriptomes of three cell types derived from common sympathoadrenal progenitors, but exhibiting variable responsiveness to acute hypoxia: CB and AM cells which are O2‐sensitive (glomus cells > chromaffin cells) and superior cervical ganglion (SCG) neurons which are practically O2‐insensitive. In the O2‐sensitive cells, we found a characteristic mRNA expression pattern of prolyl hydroxylase 3/hypoxia inducible factor 2α and up‐regulation of several genes, in particular three atypical mitochondrial electron transport subunits and some ion channels. In addition, we found that pyruvate carboxylase, an enzyme fundamental to tricarboxylic acid cycle anaplerosis, is overexpressed in CB glomus cells. We also observed that the inhibition of succinate dehydrogenase impairs CB acute O2 sensing. Our data suggest that responsiveness to acute hypoxia depends on a “signature metabolic profile” in chemoreceptor cells.
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Key points
Although lipid droplets in skeletal muscle are an important energy source during endurance exercise, our understanding of lipid metabolism in this context remains incomplete.
Using transmission electron microscopy, two distinct subcellular pools of lipid droplets can be observed in skeletal muscle – one beneath the sarcolemma and the other between myofibrils.
At rest, well‐trained leg muscles of cross‐country skiers contain 4‐ to 6‐fold more lipid droplets than equally well‐trained arm muscles, with a 3‐fold higher content in type 1 than in type 2 fibres.
During exhaustive exercise, lipid droplets between the myofibrils but not those beneath the sarcolemma are utilised by both type 1 and 2 fibres.
These findings provide insight into compartmentalisation of lipid metabolism within skeletal muscle fibres.
Abstract
Although the intramyocellular lipid pool is an important energy store during prolonged exercise, our knowledge concerning its metabolism is still incomplete. Here, quantitative electron microscopy was used to examine subcellular distribution of lipid droplets in type 1 and 2 fibres of the arm and leg muscles before and after 1 h of exhaustive exercise. Intermyofibrillar lipid droplets accounted for 85–97% of the total volume fraction, while the subsarcolemmal pool made up 3–15%. Before exercise, the volume fractions of intermyofibrillar and subsarcolemmal lipid droplets were 4‐ to 6‐fold higher in leg than in arm muscles (P

Key points
Dendritic and spine calcium imaging in combination with electrophysiology in acute slices revealed that in medial intercalated cells of the amygdala:
Action potentials back‐propagate into the dendritic tree, but due to the presence of voltage‐dependent potassium channels, probably Kv4.2 channels, attenuate over distance.
A mixed population of AMPA receptors with rectifying and linear I–V relations are present at individual spines of a single neuron.
Decay kinetics and pharmacology suggest tri‐heteromeric NMDA receptors at basolateral–intercalated cell synapses. NMDA receptors are the main contributors to spine calcium entry in response to synaptic stimulation.
Calcium signals in response to low‐ and high‐frequency stimulation, and in combination with spontaneous action potentials are locally restricted to the vicinity of active spines.
Together, these data show that calcium signalling in these GABAergic neurons is tightly controlled and acts as a local signal.
Abstract
The amygdala plays a central role in fear conditioning and extinction. The medial intercalated (mITC) neurons are GABAergic cell clusters interspaced between the basolateral (BLA) and central amygdala (CeA). These neurons are thought to play a key role in fear and extinction, controlling the output of the CeA by feed‐forward inhibition. BLA to mITC cell inputs are thought to undergo synaptic plasticity, a mechanism underlying learning, which is mediated by NMDA receptor‐dependent mechanisms that require changes in cytosolic calcium. Here, we studied the electrical and calcium signalling properties of mITC neurons in GAD67‐eGFP mice using whole‐cell patch clamp recordings and two‐photon calcium imaging. We show that action potentials back‐propagate (bAP) into dendrites, and evoke calcium transients in both the shaft and the dendritic spine. However, bAP‐mediated calcium rises in the dendrites attenuate with distance due to shunting by voltage‐gated potassium channels. Glutamatergic inputs make dual component synapses on spines. At these synapses, postsynaptic AMPA receptors can have linear or rectifying I–V relationships, indicating that some synapses express GluA2‐lacking AMPA receptors. Synaptic NMDA receptors had intermediate decay kinetics, and were only partly blocked by GuN2B selective blockers, indicating these receptors are GluN1/GluN2A/GluN2B trimers. Low‐ or high‐frequency synaptic stimulation raised spine calcium, mediated by calcium influx via NMDA receptors, was locally restricted and did not invade neighbouring spines. Our results show that in mITC neurons, postsynaptic calcium is tightly controlled, and acts as a local signal.

Key points
For correct application and interpretation of cerebral autoregulation (CA) measurements in research and in clinical care, it is essential to understand differences and similarities between dynamic and steady‐state CA.
The present study found no correlation between dynamic and steady‐state CA indices in healthy older adults.
There was variability between individuals in all (steady‐state and dynamic) autoregulatory indices, ranging from low (almost absent) to highly efficient CA in this healthy population.
These findings challenge the assumption that assessment of a single CA parameter or a single set of parameters can be generalized to overall CA functioning. Therefore, depending on specific research purposes, the choice for either steady‐state or dynamic measures or both should be weighed carefully.
Abstract
The present study aimed to investigate the relationship between dynamic (dCA) and steady‐state cerebral autoregulation (sCA). In 28 healthy older adults, sCA was quantified by a linear regression slope of proportionate (%) changes in cerebrovascular resistance (CVR) in response to proportionate (%) changes in mean blood pressure (BP) induced by stepwise sodium nitroprusside (SNP) and phenylephrine (PhE) infusion. Cerebral blood flow (CBF) was measured at the internal carotid artery (ICA) and vertebral artery (VA) and CBF velocity at the middle cerebral artery (MCA). With CVR = BP/CBF, Slope‐CVRICA, Slope‐CVRVA and Slope‐CVRiMCA were derived. dCA was assessed (i) in supine rest, analysed with transfer function analysis (gain and phase) and autoregulatory index (ARI) fit from spontaneous oscillations (ARIBaseline), and (ii) with transient changes in BP using a bolus injection of SNP (ARISNP) and PhE (ARIPhE). Comparison of sCA and dCA parameters (using Pearson's r for continuous and Spearman's ρ for ordinal parameters) demonstrated a lack of linear correlations between sCA and dCA measures. However, comparisons of parameters within dCA and within sCA were correlated. For sCA slope‐CVRVA with Slope‐CVRiMCA (r = 0.45, P

Key points
Rett syndrome is a neurodevelopmental disorder caused by loss‐of‐function mutations in MECP2, the gene encoding the transcriptional regulator methyl‐CpG‐binding protein 2 (MeCP2). Mecp2 deletion in mice results in an imbalance of excitation and inhibition in hippocampal neurons, which affects ‘Hebbian’ synaptic plasticity.
We show that Mecp2‐deficient neurons also lack homeostatic synaptic plasticity, likely due to reduced levels of EEA1, a protein involved in AMPA receptor endocytosis.
Expression of EEA1 restored homeostatic synaptic plasticity in Mecp2‐deficient neurons, providing novel targets of intervention in Rett syndrome.
Abstract
Rett syndrome is a neurodevelopmental disorder caused by loss‐of‐function mutations in MECP2, the gene encoding the transcriptional regulator methyl‐CpG‐binding protein 2 (MeCP2). Deletion of Mecp2 in mice results in an imbalance of synaptic excitation and inhibition in hippocampal pyramidal neurons, which affects ‘Hebbian’ long‐term synaptic plasticity. Since the excitatory–inhibitory balance is maintained by homeostatic mechanisms, we examined the role of MeCP2 in homeostatic synaptic plasticity (HSP) at excitatory synapses. Negative feedback HSP, also known as synaptic scaling, maintains the global synaptic strength of individual neurons in response to sustained alterations in neuronal activity. Hippocampal neurons from Mecp2 knockout (KO) mice do not show the characteristic homeostatic scaling up of the amplitude of miniature excitatory postsynaptic currents (mEPSCs) and of synaptic levels of the GluA1 subunit of AMPA‐type glutamate receptors after 48 h silencing with the Na+ channel blocker tetrodotoxin. This deficit in HSP is bidirectional because Mecp2 KO neurons also failed to scale down mEPSC amplitudes and GluA1 synaptic levels after 48 h blockade of type A GABA receptor (GABAAR)‐mediated inhibition with bicuculline. Consistent with the role of synaptic trafficking of AMPA‐type of glutamate receptors in HSP, Mecp2 KO neurons have lower levels of early endosome antigen 1 (EEA1), a protein involved in AMPA‐type glutamate receptor endocytosis. In addition, expression of EEA1 in Mecp2 KO neurons reduced mEPSC amplitudes to wild‐type levels, and restored synaptic scaling down of mEPSC amplitudes after 48 h blockade of GABAAR‐mediated inhibition with bicuculline. The identification of a molecular deficit in HSP in Mecp2 KO neurons provides potentially novel targets of intervention for improving hippocampal function in Rett syndrome individuals.

Key points
Comprehensive imaging analyses of glucose transporter 4 (GLUT4) behaviour in mouse skeletal muscle was conducted.
Quantum dot‐based single molecule nanometry revealed that GLUT4 molecules in skeletal myofibres are governed by regulatory systems involving ‘static retention’ and ‘stimulus‐dependent liberation’.
Vital imaging analyses and super‐resolution microscopy‐based morphometry demonstrated that insulin liberates the GLUT4 molecule from its static state by triggering acute heterotypic endomembrane fusion arising from the very small GLUT4‐containing vesicles in skeletal myofibres.
Prior exposure to exercise‐mimetic stimuli potentiated this insulin‐responsive endomembrane fusion event involving GLUT4‐containing vesicles, suggesting that this endomembranous regulation process is a potential site related to the effects of exercise.
Abstract
Skeletal muscle is the major systemic glucose disposal site. Both insulin and exercise facilitate translocation of the glucose transporter glucose transporter 4 (GLUT4) via distinct signalling pathways and exercise also enhances insulin sensitivity. However, the trafficking mechanisms controlling GLUT4 mobilization in skeletal muscle remain poorly understood as a resuly of technical limitations. In the present study, which employs various imaging techniques on isolated skeletal myofibres, we show that one of the initial triggers of insulin‐induced GLUT4 translocation is heterotypic endomembrane fusion arising from very small static GLUT4‐containing vesicles with a subset of transferrin receptor‐containing endosomes. Importantly, pretreatment with exercise‐mimetic stimuli potentiated the susceptibility to insulin responsiveness, as indicated by these acute endomembranous activities. We also found that AS160 exhibited stripe‐like localization close to sarcomeric α‐actinin and that insulin induced a reduction of the stripe‐like localization accompanying changes in its detergent solubility. The results of the present study thus provide a conceptual framework indicating that GLUT4 protein trafficking via heterotypic fusion is a critical feature of GLUT4 translocation in skeletal muscles and also suggest that the efficacy of the endomembranous fusion process in response to insulin is involved in the benefits of exercise.

Key points
Directionality, inherent to movements, has behavioural and neuronal correlates.
Direction of vestibular stimulation determines motor learning efficiency.
Vestibulo‐ocular reflex gain–increase correlates with Purkinje cell simple spike potentiation.
The locus of neural correlates for vestibulo‐ocular reflex adaptation is paradigm specific.
Abstract
Compensatory eye movements elicited by head rotation, also known as vestibulo‐ocular reflex (VOR), can be adapted with the use of visual feedback. The cerebellum is essential for this type of movement adaptation, although its neuronal correlates remain to be clarified. In the present study, we show that the direction of vestibular input determines the magnitude of eye movement adaptation induced by mismatched visual input in mice, with larger changes during contraversive head rotation. Moreover, the location of the neural correlate of this changed behaviour depends on the type of paradigm. Gain–increase paradigms induce increased simple spike (SS) activity in ipsilateral cerebellar Purkinje cells (PC), which is in line with eye movements triggered by optogenetic PC activation. By contrast, gain–decrease paradigms do not induce changes in SS activity, indicating that the murine vestibulo‐cerebellar cortical circuitry is optimally designed to enhance ipsiversive eye movements.

Key points
Carotid body dysfunction is recognized as a cause of hypertension in a number of cardiorespiratory diseases states and has therefore been identified as a potential therapeutic target.
Purinergic transmission is an important element of the carotid body chemotransduction pathway.
We show that inhibition of ecto‐5′‐nucleotidase (CD73) in vitro reduces carotid body basal discharge and responses to hypoxia and mitochondrial inhibition.
Additionally, inhibition of CD73 in vivo decreased the hypoxic ventilatory response, reduced the hypoxia‐induced heart rate elevation and exaggerated the blood pressure decrease in response to hypoxia.
Our data show CD73 to be a novel regulator of carotid body sensory function and therefore suggest that this enzyme may offer a new target for reducing carotid body activity in selected cardiovascular diseases.
Abstract
Augmented sensory neuronal activity from the carotid body (CB) has emerged as a principal cause of hypertension in a number of cardiovascular related pathologies, including obstructive sleep apnoea, heart failure and diabetes. Development of new targets and pharmacological treatment strategies aiming to reduce CB sensory activity may thus improve outcomes in these key patient cohorts. The present study investigated whether ecto‐5′‐nucleotidase (CD73), an enzyme that generates adenosine, is functionally important in modifying CB sensory activity and cardiovascular respiratory responses to hypoxia. Inhibition of CD73 by α,β‐methylene ADP (AOPCP) in the whole CB preparation in vitro reduced basal discharge frequency by 76 ± 5% and reduced sensory activity throughout graded hypoxia. AOPCP also significantly attenuated elevations in sensory activity evoked by mitochondrial inhibition. These effects were mimicked by antagonism of adenosine receptors with 8‐(p‐sulfophenyl) theophylline. Infusion of AOPCP in vivo significantly decreased the hypoxic ventilatory response (ΔV̇E control 74 ± 6%, ΔV̇E AOPCP 64 ± 5%, P

Key points
Intravascular ATP attenuates sympathetic vasoconstriction (sympatholysis) similar to what is observed in contracting skeletal muscle of humans, and may be an important contributor to exercise hyperaemia.
Similar to exercise, ATP‐mediated vasodilatation occurs via activation of inwardly rectifying potassium channels (KIR), and synthesis of nitric oxide (NO) and prostaglandins (PG).
However, recent evidence suggests that these dilatatory pathways are not obligatory for sympatholysis during exercise; therefore, we tested the hypothesis that the ability of ATP to blunt α1‐adrenergic vasoconstriction in resting skeletal muscle would be independent of KIR, NO, PGs and Na+/K+‐ATPase activity.
Blockade of KIR channels alone or in combination with NO, PGs and Na+/K+‐ATPase significantly reduced the vasodilatatory response to ATP, although intravascular ATP maintained the ability to attenuate α1‐adrenergic vasoconstriction.
This study highlights similarities in the vascular response to ATP and exercise, and further supports a potential role of intravascular ATP in blood flow regulation during exercise in humans.
Abstract
Exercise and intravascular ATP elicit vasodilatation that is dependent on activation of inwardly rectifying potassium (KIR) channels, with a modest reliance on nitric oxide (NO) and prostaglandin (PG) synthesis. Both exercise and intravascular ATP attenuate sympathetic α‐adrenergic vasoconstriction (sympatholysis). However, KIR channels, NO, PGs and Na+/K+‐ATPase activity are not obligatory to observe sympatholysis during exercise. To further determine similarities between exercise and intravascular ATP, we tested the hypothesis that inhibition of KIR channels, NO and PG synthesis, and Na+/K+‐ATPase would not alter the ability of ATP to blunt α1‐adrenergic vasoconstriction. In healthy subjects, we measured forearm blood flow (Doppler ultrasound) and calculated changes in vascular conductance (FVC) to intra‐arterial infusion of phenylephrine (PE; α1‐agonist) during ATP or control vasodilatator infusion, before and after KIR channel inhibition alone (barium chloride; n = 7; Protocol 1); NO (l‐NMMA) and PG (ketorolac) inhibition alone, or combined NO, PGs, Na+/K+‐ATPase (ouabain) and KIR channel inhibition (n = 6; Protocol 2). ATP attenuated PE‐mediated vasoconstriction relative to adenosine (ADO) and sodium nitroprusside (SNP) (PE‐mediated ΔFVC: ATP: −16 ± 2; ADO: −38 ± 6; SNP: −59 ± 6%; P 0.05 vs. control). These findings demonstrate that intravascular ATP modulates α1‐adrenergic vasoconstriction via pathways independent of KIR channels, NO, PGs and Na+/K+‐ATPase in humans, consistent with a role for endothelium‐derived hyperpolarization in functional sympatholysis.

The effects of acute hypoxia have been widely studied, but there are few studies of transcriptional responses to hours of hypoxia in vivo, especially in hypoxia‐tolerant tissues like skeletal muscles. We used RNA‐seq to analyse gene expression in plantaris muscles while monitoring respiration, arterial blood gases, and blood glucose in mice exposed to 8% O2 for 2 or 6 h. Rapid decreases in blood gases and a slower reduction in blood glucose suggest stress, which was accompanied by widespread changes in gene expression. Early down‐regulation of genes associated with the extracellular matrix was followed by a shift to genes associated with the nuclear lumen. Most of the early down‐regulated genes had mRNA half‐lives longer than 2 h, suggesting a role for post‐transcriptional regulation. These transcriptional changes were enriched in signalling pathways in which the PI3K/Akt signalling pathway was identified as a hub. Our analyses indicated that gene targets of PI3K/Akt but not HIF were enriched in early transcriptional responses to hypoxia. Among the PI3K/Akt targets, 75% could be explained by a deactivation of ARE‐binding protein BRF1, a target of PI3K/Akt. Consistent decreases in the phosphorylation of Akt and BRF1 were experimentally confirmed following 2 h of hypoxia. These results suggest that the PI3K/Akt signalling pathway might play a role in responses induced by acute hypoxia in skeletal muscles, partially through the de‐phosphorylation of ARE‐binding protein BRF1.
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Key points
The lower urinary tract is regulated by reflexes responsible for maintaining continence and producing efficient voiding.
It is unclear how sensory information from the bladder and urethra engages differential, state‐dependent reflexes to either maintain continence or promote voiding.
Using a new in vivo experimental approach, we quantified how sensory information from the bladder and urethra are integrated to switch reflex responses to urethral sensory feedback from maintaining continence to producing voiding.
The results demonstrate how sensory information regulates state‐dependent reflexes in the lower urinary tract and contribute to our understanding of the pathophysiology of urinary retention and incontinence where sensory feedback may engage these reflexes inappropriately.
Abstract
Lower urinary tract reflexes are mediated by peripheral afferents from the bladder (primarily in the pelvic nerve) and the urethra (in the pudendal and pelvic nerves) to maintain continence or initiate micturition. If fluid enters the urethra at low bladder volumes, reflexes relax the bladder and evoke external urethral sphincter (EUS) contraction (guarding reflex) to maintain continence. Conversely, urethral flow at high bladder volumes, excites the bladder (micturition reflex) and relaxes the EUS (augmenting reflex). We conducted measurements in a urethane‐anaesthetized in vivo rat preparation to characterize systematically the reflexes evoked by fluid flow through the urethra. We used a novel preparation to manipulate sensory feedback from the bladder and urethra independently by controlling bladder volume and urethral flow. We found a distinct bladder volume threshold (74% of bladder capacity) above which flow‐evoked bladder contractions were 252% larger and evoked phasic EUS activation 2.6 times as often as responses below threshold, clearly demonstrating a discrete transition between continence (guarding) and micturition (augmenting) reflexes. Below this threshold urethral flow evoked tonic EUS activity, indicative of the guarding reflex, that was proportional to the urethral flow rate. These results demonstrate the complementary roles of sensory feedback from the bladder and urethra in regulating reflexes in the lower urinary tract that depend on the state of the bladder. Understanding the neural control of functional reflexes and how they are mediated by sensory information in the bladder and urethra will open new opportunities, especially in neuromodulation, to treat pathologies of the lower urinary tract.

Key points
Neuronal nicotinic acetylcholine receptors (nAChRs) play a fundamental role in the attentional circuitry throughout the mammalian CNS.
In the present study, we report a novel finding that ageing negatively impacts nAChR efficacy in auditory thalamus, and this is probably the result of a loss of nAChR density (Bmax) and changes in the subunit composition of nAChRs.
Our data support the hypothesis that age‐related maladaptive changes involving nAChRs within thalamocortical circuits partially underpin the difficulty that elderly adults experience with respect to attending to speech and other salient acoustic signals.
Abstract
The flow of auditory information through the medial geniculate body (MGB) is regulated, in part, by cholinergic projections from the pontomesencephalic tegmentum. The functional significance of these projections is not fully established, although they have been strongly implicated in the allocation of auditory attention. Using in vitro slice recordings, we have analysed postsynaptic function and pharmacology of neuronal nicotinic ACh receptors (nAChRs) in young adult and the aged rat MGB. We find that ACh produces significant excitatory postsynaptic actions on young MGB neurons, probably mediated by β2‐containing heteromeric nAChRs. Radioligand binding studies show a significant age‐related loss of heteromeric nAChR receptor number, which supports patch clamp data showing an age‐related loss in ACh efficacy in evoking postsynaptic responses. Use of the β2‐selective nAChR antagonist, dihydro‐β‐erythroidine, suggests that loss of cholinergic efficacy may also be the result of an age‐related subunit switch from high affinity β2‐containing nAChRs to low affinity β4‐containing nAChRs, in addition to the loss of total nAChR number. This age‐related nAChR dysfunction may partially underpin the attentional deficits that contribute to the loss of speech understanding in the elderly.

Key points
At rat calyx of Held terminals, ATP was required not only for slow endocytosis, but also for rapid phase of compensatory endocytosis.
An ATP‐independent form of endocytosis was recruited to accelerate membrane retrieval at increased activity and temperature.
ATP‐independent endocytosis primarily involved retrieval of pre‐existing membrane, which depended on Ca2+ and the activity of neutral sphingomyelinase but not clathrin‐coated pit maturation.
ATP‐independent endocytosis represents a non‐canonical mechanism that can efficiently retrieve membrane at physiological conditions without competing for the limited ATP at elevated neuronal activity.
Abstract
Neurotransmission relies on membrane endocytosis to maintain vesicle supply and membrane stability. Endocytosis has been generally recognized as a major ATP‐dependent function, which efficiently retrieves more membrane at elevated neuronal activity when ATP consumption within nerve terminals increases drastically. This paradox raises the interesting question of whether increased activity recruits ATP‐independent mechanism(s) to accelerate endocytosis at the same time as preserving ATP availability for other tasks. To address this issue, we studied ATP requirement in three typical forms of endocytosis at rat calyx of Held terminals by whole‐cell membrane capacitance measurements. At room temperature, blocking ATP hydrolysis effectively abolished slow endocytosis and rapid endocytosis but only partially inhibited excess endocytosis following intense stimulation. The ATP‐independent endocytosis occurred at calyces from postnatal days 8–15, suggesting its existence before and after hearing onset. This endocytosis was not affected by a reduction of exocytosis using the light chain of botulinum toxin C, nor by block of clathrin‐coat maturation. It was abolished by EGTA, which preferentially blocked endocytosis of retrievable membrane pre‐existing at the surface, and was impaired by oxidation of cholesterol and inhibition of neutral sphingomyelinase. ATP‐independent endocytosis became more significant at 34–35°C, and recovered membrane by an amount that, on average, was close to exocytosis. The results of the present study suggest that activity and temperature recruit ATP‐independent endocytosis of pre‐existing membrane (in addition to ATP‐dependent endocytosis) to efficiently retrieve membrane at nerve terminals. This less understood endocytosis represents a non‐canonical mechanism regulated by lipids such as cholesterol and sphingomyelinase.

Key points
The present study demonstrates that electromyograms (EMGs) obtained during locomotor activity in mice were effective for identification of early physiological markers of amyotrophic lateral sclerosis (ALS). These measures could be used to evaluate therapeutic intervention strategies in animal models of ALS.
Several parameters of locomotor activity were shifted early in the disease time course in SOD1G93A mice, especially when the treadmill was inclined, including intermuscular phase, burst skew and amplitude of the locomotor bursts.
The results of the present study indicate that early compensatory changes may be taking place within the neural network controlling locomotor activity, including spinal interneurons.
Locomotor EMGs could have potential use as a clinical diagnostic tool.
Abstract
To improve our understanding of early disease mechanisms and to identify reliable biomarkers of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease, we measured electromyogram (EMG) activity in hind limb muscles of SOD1G93A mice. By contrast to clinical diagnostic measures using EMGs, which are performed on quiescent patients, we monitored activity during treadmill running aiming to detect presymptomatic changes in motor patterning. Chronic EMG electrodes were implanted into vastus lateralis, biceps femoris posterior, lateral gastrocnemius and tibialis anterior in mice from postnatal day 55 to 100 and the results obtained were assessed using linear mixed models. We evaluated differences in parameters related to EMG amplitude (peak and area) and timing (phase and skew, a measure of burst shape) when animals ran on level and inclined treadmills. There were significant changes in both the timing of activity and the amplitude of EMG bursts in SOD1G93A mice. Significant differences between wild‐type and SOD1G93A mice were mainly observed when animals locomoted on inclined treadmills. All muscles had significant effects of mutation that were independent of age. These novel results indicate (i) locomotor EMG activity might be an early measure of disease onset; (ii) alterations in locomotor patterning may reflect changes in neuronal drive and compensation at the network level including altered activity of spinal interneurons; and (iii) the increased power output necessary on an inclined treadmill was important in revealing altered activity in SOD1G93A mice.

Key points
With the development of the metabolic syndrome, both post‐capillary and collecting venular dilator reactivity within the skeletal muscle of obese Zucker rats (OZR) is impaired.
The impaired dilator reactivity in OZR reflects a loss in venular nitric oxide and PGI2 bioavailability, associated with the chronic elevation in oxidant stress.
Additionally, with the impaired dilator responses, a modest increase in adrenergic constriction combined with an elevated thromboxane A2 production may contribute to impaired functional dilator and hyperaemic responses at the venular level.
For the shift in skeletal muscle venular function with development of the metabolic syndrome, issues such as aggregate microvascular perfusion resistance, mass transport and exchange within with capillary networks, and fluid handling across the microcirculation are compelling avenues for future investigation.
Abstract
While research into vascular outcomes of the metabolic syndrome has focused on arterial/arteriolar and capillary levels, investigation into venular function and how this impacts responses has received little attention. Using the in situ cremaster muscle of obese Zucker rats (OZR; with lean Zucker rats (LZR) as controls), we determined indices of venular function. At ∼17 weeks of age, skeletal muscle post‐capillary venular density was reduced by ∼20% in LZR vs. OZR, although there was no evidence of remodelling of the venular wall. Venular tone at ∼25 μm (post‐capillary) and ∼75 μm (collecting) diameter was elevated in OZR vs. LZR. Venular dilatation to acetylcholine was blunted in OZR vs. LZR due to increased oxidant stress‐based loss of nitric oxide bioavailability (post‐capillary) and increased α1‐ (and α2‐) mediated constrictor tone (collecting). Venular constrictor responses in OZR were comparable to LZR for most stimuli, although constriction to α1‐adrenoreceptor stimulation was elevated. In response to field stimulation of the cremaster muscle (0.5, 1, 3 Hz), venular dilator and hyperaemic responses to lower frequencies were blunted in OZR, but responses at 3 Hz were similar between strains. Venous production of TxA2 was higher in OZR than LZR and significantly higher than PGI2 production in either following arachidonic acid challenge. These results suggest that multi‐faceted alterations to skeletal muscle venular function in OZR may contribute to alterations in upstream capillary pressure profiles and the transcapillary exchange of solutes and water under conditions of metabolic syndrome.

Glutamatergic signalling is critical in the nucleus tractus solitarii (nTS) for cardiorespiratory homeostasis and initiation of sensory reflexes, including the chemoreflex activated during hypoxia. Maintenance of nTS glutamate concentration occurs in part through astrocytic excitatory amino acid transporters (EAATs). We previously established the importance of EAATs in the nTS by demonstrating their inhibition produced neuronal excitation to alter basal cardiorespiratory function. Since EAAT2 is the most expressed EAAT in the nTS, this study specifically determined EAAT2's role in nTS astrocytes, their influence on neuronal and synaptic properties, and ultimately on basal and reflex cardiorespiratory function. The EAAT2 specific antagonist dihydrokainate (DHK) was microinjected into the anaesthetized rat nTS or applied to rat nTS slices. DHK produced depressor, bradycardic and sympathoinhibitory responses and reduced neural respiration in the intact rat, mimicking responses to glutamate excitation. DHK also enhanced responses to glutamate microinjection. DHK elevated extracellular nTS glutamate concentration, depolarized neurons and enhanced spontaneous EPSCs. EAAT2 block also augmented action potential discharge in chemosensitive nTS neurons. Glial recordings confirmed EAAT2 is functional on nTS astrocytes. Neuronal excitation and cardiorespiratory effects following EAAT2 inhibition were due to activation of putative extrasynaptic AMPA receptors as their antagonism blocked DHK responses in the intact rat nTS and the slice. The DHK‐induced elevation of extracellular glutamate and neuronal excitation augmented chemoreflex‐mediated pressor, sympathoexcitatory and minute neural ventilation responses in the rat. These data shed new light on the important role astrocytic EAAT2 plays on buffering nTS excitation and overall cardiorespiratory function.
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Several neocortical projections converge onto the basolateral complex of the amygdala (BLA), including the lateral orbitofrontal cortex (lOFC) and medial prefrontal cortex (mPFC). Lateral orbitofrontal input to BLA is important for cue‐outcome contingencies, while medial prefrontal input is essential for emotion control. In this study, we examined how mPFC, specifically the infralimbic (IL) division of mPFC, modulates the lOFC‐BLA information flow, using combined in vivo extracellular single‐unit recordings and pharmacological manipulations in anesthetized rats. We found that the majority (over 95%) of BLA neurons that responded to lOFC stimulation also responded to mPFC stimulation. Compared to basal condition, pharmacological (N‐Methyl‐D‐aspartate, NMDA) or electrical activation of the mPFC exerted an inhibitory modulation of the lOFC‐BLA pathway, which was reversed with intra‐amygdala blockade of GABAergic receptors with combined GABAA and GABAB antagonists (bicuculline and saclofen). Moreover, mPFC tetanus potentiated the lOFC‐BLA pathway, but mPFC tetanus or low‐frequency stimulation (LFS) did not alter its inhibitory modulatory gating on the lOFC‐BLA pathway. These results show that the mPFC potently inhibits lOFC drive of BLA neurons in a GABA‐dependent manner. Our result is informative in understanding the normal and potential pathophysiological state of emotion and contingency associations regulating behaviour.
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The hypoxic ventilatory response (HVR) is biphasic, consisting of a phase I increase in ventilation followed by a secondary depression (to a steady‐state phase II) that can be life‐threatening in premature infants who suffer from frequent apneas and respiratory depression. ATP released in the ventrolateral medulla oblongata during hypoxia attenuates the secondary depression. We explored a working hypothesis that vesicular release of ATP by astrocytes in the preBötzinger Complex (preBötC) inspiratory rhythm generating network acts via P2Y1 receptors to mediate this effect. Blockade of vesicular exocytosis in preBötC astrocytes bilaterally (using an adenoviral vector to specifically express tetanus toxin light chain in astrocytes) reduced the HVR in anaesthetized rats, indicating that exocytotic release of a gliotransmitter within the preBötC contributes to the hypoxia‐induced increases in ventilation. Unilateral blockade of P2Y1 receptors in the preBötC via local antagonist injection enhanced the secondary respiratory depression, suggesting that a significant component of the phase II increase in ventilation is mediated by ATP acting at P2Y1 receptors. In vitro responses of the preBötC inspiratory network, preBötC inspiratory neurons and cultured preBötC glia to purinergic agents demonstrated that the P2Y1 receptor‐mediated increase in fictive inspiratory frequency involves Ca2+ recruitment from intracellular stores leading to increases in intracellular Ca2+ ([Ca2+]i) in inspiratory neurons and glia. These data suggest that ATP is released by preBötC astrocytes during hypoxia and acts via P2Y1 receptors on inspiratory neurons (and/or glia) to evoke Ca2+ release from intracellular stores and an increase in ventilation that counteracts the hypoxic respiratory depression.
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Key points
Mechanical signals play a critical role in the regulation of muscle mass, but the molecules that sense mechanical signals and convert this stimulus into the biochemical events that regulate muscle mass remain ill‐defined.
Here we report a mass spectrometry‐based workflow to study the changes in protein phosphorylation that occur in mouse skeletal muscle 1 h after a bout of electrically evoked maximal‐intensity contractions (MICs).
Our dataset provides the first comprehensive map of the MIC‐regulated phosphoproteome.
Using unbiased bioinformatics approaches, we demonstrate that our dataset leads to the identification of many well‐known MIC‐regulated signalling pathways, as well as to a plethora of novel MIC‐regulated events.
We expect that our dataset will serve as a fundamentally important resource for muscle biologists, and help to lay the foundation for entirely new hypotheses in the field.
Abstract
The maintenance of skeletal muscle mass is essential for health and quality of life. It is well recognized that maximal‐intensity contractions, such as those which occur during resistance exercise, promote an increase in muscle mass. Yet, the molecules that sense the mechanical information and convert it into the signalling events (e.g. phosphorylation) that drive the increase in muscle mass remain undefined. Here we describe a phosphoproteomics workflow to examine the effects of electrically evoked maximal‐intensity contractions (MICs) on protein phosphorylation in mouse skeletal muscle. While a preliminary phosphoproteomics experiment successfully identified a number of MIC‐regulated phosphorylation events, a large proportion of these identifications were present on highly abundant myofibrillar proteins. We subsequently incorporated a centrifugation‐based fractionation step to deplete the highly abundant myofibrillar proteins and performed a second phosphoproteomics experiment. In total, we identified 5983 unique phosphorylation sites of which 663 were found to be regulated by MIC. GO term enrichment, phosphorylation motif analyses, and kinase‐substrate predictions indicated that the MIC‐regulated phosphorylation sites were chiefly modified by mTOR, as well as multiple isoforms of the MAPKs and CAMKs. Moreover, a high proportion of the regulated phosphorylation sites were found on proteins that are associated with the Z‐disc, with over 74% of the Z‐disc proteins experiencing robust changes in phosphorylation. Finally, our analyses revealed that the phosphorylation state of two Z‐disc kinases (striated muscle‐specific serine/threonine protein kinase and obscurin) was dramatically altered by MIC, and we propose ways these kinases could play a fundamental role in skeletal muscle mechanotransduction.

Key points
Progressive depletion of all vascular endothelial growth factor A (VEGF‐A) splice isoforms from the kidney results in proteinuria and increased glomerular water permeability, which are both rescued by over‐expression of VEGF‐A165b only.
VEGF‐A165b rescues the increase in glomerular basement membrane and podocyte slit width, as well as the decrease in sub‐podocyte space coverage, produced by VEGF‐A depletion.
VEGF‐A165b restores the expression of platelet endothelial cell adhesion molecule in glomerular endothelial cells and glomerular capillary circumference.
VEGF‐A165b has opposite effects to VEGF‐A165 on the expression of genes involved in endothelial cell migration and proliferation.
Abstract
Chronic kidney disease is strongly associated with a decrease in the expression of vascular endothelial growth factor A (VEGF‐A). However, little is known about the contribution of VEGF‐A splice isoforms to kidney physiology and pathology. Previous studies suggest that the splice isoform VEGF‐A165b (resulting from alternative usage of a 3′ splice site in the terminal exon) is protective for kidney function. In the present study, we show, in a quad‐transgenic model, that over‐expression of VEGF‐A165b alone is sufficient to rescue the increase in proteinuria, as well as glomerular water permeability, in the context of progressive depletion of all VEGF‐A isoforms from the podocytes. Ultrastructural studies show that the glomerular basement membrane is thickened, podocyte slit width is increased and sub‐podocyte space coverage is reduced when VEGF‐A is depleted, all of which are rescued in VEGF‐A165b over‐expressors. VEGF‐A165b restores the expression of platelet endothelial cell adhesion molecule‐1 in glomerular endothelial cells and glomerular capillary circumference. Mechanistically, it increases VEGF receptor 2 expression both in vivo and in vitro and down‐regulates genes involved in migration and proliferation of endothelial cells, otherwise up‐regulated by the canonical isoform VEGF‐A165. The results of the present study indicate that manipulation of VEGF‐A splice isoforms could be a novel therapeutic avenue in chronic glomerular disease.

Key points
Ageing‐induced endothelial dysfunction contributes to organ dysfunction and progression of cardiovascular disease. VE‐cadherin clustering at adherens junctions promotes protective endothelial functions, including endothelium‐dependent dilatation.
Ageing increased internalization and degradation of VE‐cadherin, resulting in impaired activity of adherens junctions.
Inhibition of VE‐cadherin clustering at adherens junctions (function‐blocking antibody; FBA) reduced endothelial dilatation in young arteries but did not affect the already impaired dilatation in old arteries. After junctional disruption with the FBA, dilatation was similar in young and old arteries.
Src tyrosine kinase activity and tyrosine phosphorylation of VE‐cadherin were increased in old arteries. Src inhibition increased VE‐cadherin at adherens junctions and increased endothelial dilatation in old, but not young, arteries. Src inhibition did not increase dilatation in old arteries treated with the VE‐cadherin FBA.
Ageing impairs the activity of adherens junctions, which contributes to endothelial dilator dysfunction. Restoring the activity of adherens junctions could be of therapeutic benefit in vascular ageing.
Abstract
Endothelial dilator dysfunction contributes to pathological vascular ageing. Experiments assessed whether altered activity of endothelial adherens junctions (AJs) might contribute to this dysfunction. Aortas and tail arteries were isolated from young (3–4 months) and old (22–24 months) F344 rats. VE‐cadherin immunofluorescent staining at endothelial AJs and AJ width were reduced in old compared to young arteries. A 140 kDa VE‐cadherin species was present on the cell surface and in TTX‐insoluble fractions, consistent with junctional localization. Levels of the 140 kDa VE‐cadherin were decreased, whereas levels of a TTX‐soluble 115 kDa VE‐cadherin species were increased in old compared to young arteries. Acetylcholine caused endothelium‐dependent dilatation that was decreased in old compared to young arteries. Disruption of VE‐cadherin clustering at AJs (function‐blocking antibody, FBA) inhibited dilatation to acetylcholine in young, but not old, arteries. After the FBA, there was no longer any difference in dilatation between old and young arteries. Src activity and tyrosine phosphorylation of VE‐cadherin were increased in old compared to young arteries. In old arteries, Src inhibition (saracatinib) increased: (i) 140 kDa VE‐cadherin in the TTX‐insoluble fraction, (ii) VE‐cadherin intensity at AJs, (iii) AJ width, and (iv) acetylcholine dilatation. In old arteries treated with the FBA, saracatinib no longer increased acetylcholine dilatation. Saracatinib did not affect dilatation in young arteries. Therefore, ageing impairs AJ activity, which appears to reflect Src‐induced phosphorylation, internalization and degradation of VE‐cadherin. Moreover, impaired AJ activity can account for the endothelial dilator dysfunction in old arteries. Restoring endothelial AJ activity may be a novel therapeutic approach to vascular ageing.

Key points
Learning and memory storage requires neuronal plasticity induced in the hippocampus and other related brain areas, and this process is thought to rely on synchronized activity in neural networks.
We used paired whole‐cell recording in vivo to examine the synchronized activity that was induced in hippocampal CA1 neurons by associative fear learning.
We found that both membrane potential synchronization and spike synchronization of CA1 neurons could be transiently enhanced after task learning, as observed on day 1 but not day 5.
On day 1 after learning, CA1 neurons showed a decrease in firing threshold and rise times of suprathreshold membrane potential changes as well as an increase in spontaneous firing rates, possibly contributing to the enhancement of spike synchronization.
The transient enhancement of CA1 neuronal synchronization may play important roles in the induction of neuronal plasticity for initial storage and consolidation of associative memory.
Abstract
The hippocampus is critical for memory acquisition and consolidation. This function requires activity‐ and experience‐induced neuronal plasticity. It is known that neuronal plasticity is largely dependent on synchronized activity. As has been well characterized, repetitive correlated activity of presynaptic and postsynaptic neurons can lead to long‐term modifications at their synapses. Studies on network activity have also suggested that memory processing in the hippocampus may involve learning‐induced changes of neuronal synchronization, as observed in vivo between hippocampal CA3 and CA1 networks as well as between the rhinal cortex and the hippocampus. However, further investigation of learning‐induced synchronized activity in the hippocampus is needed for a full understanding of hippocampal memory processing. In this study, by performing paired whole‐cell recording in vivo on CA1 pyramidal cells (PCs) in anaesthetized adult rats, we examined CA1 neuronal synchronization before and after associative fear learning. We first found in naive animals that there was a low level of membrane potential (MP) synchronization and spike synchronization of CA1 PCs. In conditioned animals, we found a significant enhancement of both MP synchronization and spike synchronization, as observed on day 1 after learning, and this enhancement was transient and not observed on day 5. Accompanying learning‐induced synchronized activity was a decreased firing threshold and rise time of suprathreshold MP changes as well as an increased spontaneous firing rate, possibly contributing to the enhanced spike synchronization. The transiently enhanced CA1 neuronal synchronization may have important roles in generating neuronal plasticity for hippocampal storage and consolidation of associative memory traces.

Key points
Sudden unloading of preloaded wrist muscles elicits motion to a new wrist position. Such motion is prevented if subjects unload muscles using the contralateral arm (self‐unloading).
Corticospinal influences originated from the primary motor cortex maintain tonic influences on motoneurons of wrist muscles before sudden unloading but modify these influences prior to the onset and until the end of self‐unloading.
Results are interpreted based on the previous finding that intentional actions are caused by central, particularly corticospinal, shifts in the spatial thresholds at which wrist motoneurons are activated, thus predetermining the attractor point at which the neuromuscular periphery achieves mechanical balance with environment forces.
By maintaining or shifting the thresholds, descending systems let body segments go to the equilibrium position in the respective unloading tasks without the pre‐programming of kinematics or muscle activation patterns.
The study advances the understanding of how motor actions in general, and anticipation in particular, are controlled.
Abstract
The role of corticospinal (CS) pathways in anticipatory motor actions was evaluated using transcranial magnetic stimulation (TMS) of the primary motor cortex projecting to motoneurons (MNs) of wrist muscles. Preloaded wrist flexors were suddenly unloaded by the experimenter or by the subject using the other hand (self‐unloading). After sudden unloading, the wrist joint involuntarily flexed to a new position. In contrast, during self‐unloading the wrist remained almost motionless, implying that an anticipatory postural adjustment occurred. In the self‐unloading task, anticipation was manifested by a decrease in descending facilitation of pre‐activated flexor MNs starting ∼72 ms before changes in the background EMG activity. Descending facilitation of extensor MNs began to increase ∼61 ms later. Conversely, these influences remained unchanged before sudden unloading, implying the absence of anticipation. We also tested TMS responses during EMG silent periods produced by brief muscle shortening, transiently resulting in similar EMG levels before the onset and after the end of self‐unloading. We found reduced descending facilitation of flexor MNs after self‐unloading. To explain why the wrist excursion was minimized in self‐unloading due to these changes in descending influences, we relied on previous demonstrations that descending systems pre‐set the threshold positions of body segments at which muscles begin to be activated, thus predetermining the equilibrium point to which the system is attracted. Based on this notion, a more consistent explanation of the kinematic, EMG and descending patterns in the two types of unloading is proposed compared to the alternative notion of direct pre‐programming of kinematic and/or EMG patterns.

Key points
The highly variable phenotypes observed in patients with classic Bartter's syndrome (BS) remain unsatisfactorily explained. The wide spectrum of functional severity of CLCNKB mutations may contribute to the phenotypic variability, and the genotype–phenotype association has not been established.
Low‐level expression of the human ClC‐Kb channel in mammalian cells impedes the functional study of CLCNKB mutations, and the underlying cause is still unclear.
The human ClC‐Kb channel is highly degraded by proteasome in human embryonic kidney cells. The C‐terminal in‐frame green fluorescent protein fusion may slow down the proteasome‐mediated proteolysis. Barttin co‐expression necessarily improves the stability, membrane trafficking and gating of ClC‐Kb.
CLCNKB mutations in barttin‐binding sites, dimer interface or selectivity filter often have severe functional consequences.
The remaining chloride conductance of the ClC‐Kb mutant channel significantly correlates with the phenotypes, such as age at diagnosis, plasma chloride concentration, and the degree of calciuria in patients with classic BS.
Abstract
Mutations in the CLCNKB gene encoding the human voltage‐gated chloride ClC‐Kb (hClC‐Kb) channel cause classic Bartter's syndrome (BS). In contrast to antenatal BS, classic BS manifests with highly variable phenotypes. The functional severity of the mutant channel has been proposed to explain this phenomenon. Due to difficulties in the expression of hClC‐Kb in heterologous expression systems, the functional consequences of mutant channels have not been thoroughly examined, and the genotype–phenotype association has not been established. In this study, we found that hClC‐Kb, when expressed in human embryonic kidney (HEK) cells, was unstable due to degradation by proteasome. In‐frame fusion of green fluorescent protein (GFP) to the C‐terminus of the channel may ameliorate proteasome degradation. Co‐expression of barttin increased protein abundance and membrane trafficking of hClC‐Kb and markedly increased functional chloride current. We then functionally characterized 18 missense mutations identified in our classic BS cohort and others using HEK cells expressing hClC‐Kb‐GFP. Most CLCNKB mutations resulted in marked reduction in protein abundance and chloride current, especially those residing at barttin binding sites, dimer interface and selectivity filter. We enrolled classic BS patients carrying homozygous missense mutations with well‐described functional consequences and clinical presentations for genotype–phenotype analysis. We found significant correlations of mutant chloride current with the age at diagnosis, plasma chloride concentration and urine calcium excretion rate. In conclusion, hClC‐Kb expression in HEK cells is susceptible to proteasome degradation, and fusion of GFP to the C‐terminus of hClC‐Kb improves protein expression. The functional severity of the CLCNKB mutation is an important determinant of the phenotype in classic BS.

Key points
The lipid droplet (LD)‐associated perilipin (PLIN) proteins promote intramuscular triglyceride (IMTG) storage, although whether the abundance and association of the PLIN proteins with LDs is related to the diverse lipid storage in muscle between trained and sedentary individuals is unknown.
We show that lipid infusion augments IMTG content in type I fibres of both trained and sedentary individuals. Most importantly, despite there being no change in PLIN protein content, lipid infusion did increase the number of LDs connected with PLIN proteins in trained individuals only.
We conclude that trained individuals are able to redistribute the pre‐existing pool of PLIN proteins to an expanded LD pool during lipid infusion and, via this adaptation, may support the storage of fatty acids in IMTG.
Abstract
Because the lipid droplet (LD)‐associated perilipin (PLIN) proteins promote intramuscular triglyceride (IMTG) storage, we investigated the hypothesis that differential protein content of PLINs and their distribution with LDs may be linked to the diverse lipid storage in muscle between trained and sedentary individuals. Trained (n = 11) and sedentary (n = 10) subjects, matched for age, sex and body mass index, received either a 6 h lipid or glycerol infusion in the setting of a concurrent hyperinsulinaemic–euglycaemic clamp. Sequential muscle biopsies (0, 2 and 6 h) were analysed using confocal immunofluorescence microscopy for fibre type‐specific IMTG content and PLIN associations with LDs. In both groups, lipid infusion increased IMTG content in type I fibres (trained: +62%, sedentary: +79%; P

During functional spinal neural network activity motoneurons receive intense synaptic input, and this could modulate the threshold for action potential generation, providing the ability to dynamically adjust the excitability and recruitment order for functional needs. In the present study we investigated the dynamics of action potential threshold during motor network activity. Intracellular recordings from spinal motoneurons in an ex vivo carapace‐spinal cord preparation from adult turtles were performed during two distinct types of motor behaviour – fictive scratching and fictive swimming. We found that the threshold of the first spike in episodes of scratching and swimming was the lowest. The threshold potential depolarizes by about 10 mV within each burst of spikes generated during scratch and swim network activity and recovers between bursts to a slightly depolarized level. Depolarization of the threshold potential results in decreased excitability of motoneurons. Synaptic inputs do not modulate the threshold of the first action potential during episodes of scratching or of swimming. There is no correlation between changes in spike threshold and interspike intervals within bursts. Slow synaptic integration that results in a wave of membrane potential depolarization rather than fast synaptic events preceding each spike is the factor influencing the threshold potential within firing bursts during motor behaviours.
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Key points
Cerebellar Purkinje cells (PCs) generate two types of action potentials, simple and complex spikes. Although they are generated by distinct mechanisms, interactions between the two spike types exist.
Zebrin staining produces alternating positive and negative stripes of PCs across most of the cerebellar cortex. Thus, here we compared simple spike–complex spike interactions both within and across zebrin populations.
Simple spike activity undergoes a complex modulation preceding and following a complex spike. The amplitudes of the pre‐ and post‐complex spike modulation phases were correlated across PCs. On average, the modulation was larger for PCs in zebrin positive regions.
Correlations between aspects of the complex spike waveform and simple spike activity were found, some of which varied between zebrin positive and negative PCs.
The implications of the results are discussed with regard to hypotheses that complex spikes are triggered by rises in simple spike activity for either motor learning or homeostatic functions.
Abstract
Purkinje cells (PCs) generate two types of action potentials, called simple and complex spikes (SSs and CSs). We first investigated the CS‐associated modulation of SS activity and its relationship to the zebrin status of the PC. The modulation pattern consisted of a pre‐CS rise in SS activity, and then, following the CS, a pause, a rebound, and finally a late inhibition of SS activity for both zebrin positive (Z+) and negative (Z−) cells, though the amplitudes of the phases were larger in Z+ cells. Moreover, the amplitudes of the pre‐CS rise with the late inhibitory phase of the modulation were correlated across PCs. In contrast, correlations between modulation phases across CSs of individual PCs were generally weak. Next, the relationship between CS spikelets and SS activity was investigated. The number of spikelets/CS correlated with the average SS firing rate only for Z+ cells. In contrast, correlations across CSs between spikelet numbers and the amplitudes of the SS modulation phases were generally weak. Division of spikelets into likely axonally propagated and non‐propagated groups (based on their interspikelet interval) showed that the correlation of spikelet number with SS firing rate primarily reflected a relationship with non‐propagated spikelets. In sum, the results show both zebrin‐related and non‐zebrin‐related physiological heterogeneity in SS–CS interactions among PCs, which suggests that the cerebellar cortex is more functionally diverse than is assumed by standard theories of cerebellar function.

Key points
Dysferlin, the protein missing in limb girdle muscular dystrophy 2B and Miyoshi myopathy, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage‐induced Ca2+ transients against loss after osmotic shock injury (OSI).
Local expression of dysferlin in dysferlin‐null myofibres increases transient amplitude to control levels and protects them from loss after OSI.
Inhibitors of ryanodine receptors (RyR1) and L‐type Ca2+ channels protect voltage‐induced Ca2+ transients from loss; thus both proteins play a role in injury in dysferlin's absence. Effects of Ca2+‐free medium and S107, which inhibits SR Ca2+ leak, suggest the SR as the primary source of Ca2+ responsible for the loss of the Ca2+ transient upon injury.
Ca2+ waves were induced by OSI and suppressed by exogenous dysferlin.
We conclude that dysferlin prevents injury‐induced SR Ca2+ leak.
Abstract
Dysferlin concentrates in the transverse tubules of skeletal muscle and stabilizes Ca2+ transients when muscle fibres are subjected to osmotic shock injury (OSI). We show here that voltage‐induced Ca2+ transients elicited in dysferlin‐null A/J myofibres were smaller than control A/WySnJ fibres. Regional expression of Venus‐dysferlin chimeras in A/J fibres restored the full amplitude of the Ca2+ transients and protected against OSI. We also show that drugs that target ryanodine receptors (RyR1: dantrolene, tetracaine, S107) and L‐type Ca2+ channels (LTCCs: nifedipine, verapamil, diltiazem) prevented the decrease in Ca2+ transients in A/J fibres following OSI. Diltiazem specifically increased transients by ∼20% in uninjured A/J fibres, restoring them to control values. The fact that both RyR1s and LTCCs were involved in OSI‐induced damage suggests that damage is mediated by increased Ca2+ leak from the sarcoplasmic reticulum (SR) through the RyR1. Congruent with this, injured A/J fibres produced Ca2+ sparks and Ca2+ waves. S107 (a stabilizer of RyR1–FK506 binding protein coupling that reduces Ca2+ leak) or local expression of Venus‐dysferlin prevented OSI‐induced Ca2+ waves. Our data suggest that dysferlin modulates SR Ca2+ release in skeletal muscle, and that in its absence OSI causes increased RyR1‐mediated Ca2+ leak from the SR into the cytoplasm.

Key points
Muscular contractions performed using a combination of low external loads and partial restriction of limb blood flow appear to induce substantial gains in muscle strength and muscle mass.
This exercise regime may initially induce muscular stress and damage; however, the effects of a period of blood flow restricted training on these parameters remain largely unknown.
The present study shows that short‐term, high‐frequency, low‐load muscle training performed with partial blood flow restriction does not induce significant muscular damage.
However, signs of myocellular stress and inflammation that were observed in the early phase of training and after the training intervention, respectively, may be facilitating the previously reported gains in myogenic satellite cell content and muscle hypertrophy.
The present results improve our current knowledge about the physiological effects of low‐load muscular contractions performed under blood flow restriction and may provide important information of relevance for future therapeutic treatment of muscular atrophy.
Abstract
Previous studies indicate that low‐load muscle contractions performed under local blood flow restriction (BFR) may initially induce muscle damage and stress. However, whether these factors are evoked with longitudinal BFR training remains unexplored at the myocellular level. Two distinct study protocols were conducted, covering 3 weeks (3 wk) or one week (1 wk). Subjects performed BFR exercise (100 mmHg, 20% 1RM) to concentric failure (BFRE) (3 wk/1 wk), while controls performed work‐matched (LLE) (3 wk) or high‐load (HLE; 70% 1RM) (1 wk) free‐flow exercise. Muscle biopsies (3 wk) were obtained at baseline (Pre), 8 days into the intervention (Mid8), and 3 and 10 days after training cessation (Post3, Post10) to examine macrophage (M1/M2) content as well as heat shock protein (HSP27/70) and tenascin‐C expression. Blood samples (1 wk) were collected before and after (0.1–24 h) the first and last training session to examine markers of muscle damage (creatine kinase), oxidative stress (total antibody capacity, glutathione) and inflammation (monocyte chemotactic protein‐1, interleukin‐6, tumour necrosis factor α). M1‐macrophage content increased 108–165% with BFRE and LLE at Post3 (P

Key points
The nucleus accumbens (nAc) is involved in addiction‐related behaviour caused by several drugs of abuse, including alcohol.
Glycine receptors (GlyRs) are potentiated by ethanol and they have been implicated in the regulation of accumbal dopamine levels.
We investigated the presence of GlyR subunits in nAc and their modulation by ethanol in medium spiny neurons (MSNs) of the mouse nAc.
We found that the GlyR α1 subunit is preferentially expressed in nAc and is potentiated by ethanol.
Our study shows that GlyR α1 in nAc is a new target for development of novel pharmacological tools for behavioural intervention in drug abuse.
Abstract
Alcohol abuse causes major social, economic and health‐related problems worldwide. Alcohol, like other drugs of abuse, increases levels of dopamine in the nucleus accumbens (nAc), facilitating behavioural reinforcement and substance abuse. Previous studies suggested that glycine receptors (GlyRs) are involved in the regulation of accumbal dopamine levels. Here, we investigated the presence of GlyRs in accumbal dopamine receptor medium spiny neurons (MSNs) of C57BL/6J mice, analysing mRNA expression levels and immunoreactivity of GlyR subunits, as well as ethanol sensitivity. We found that GlyR α1 subunits are expressed at higher levels than α2, α3 and β in the mouse nAc and were located preferentially in dopamine receptor 1 (DRD1)‐positive MSNs. Interestingly, the glycine‐evoked currents in dissociated DRD1‐positive MSNs were potentiated by ethanol. Also, the potentiation of the GlyR‐mediated tonic current by ethanol suggests that they modulate the excitability of DRD1‐positive MSNs in nAc. This study should contribute to understanding the role of GlyR α1 in the reward system and might help to develop novel pharmacological therapies to treat alcoholism and other addiction‐related and compulsive behaviours.

The neurovascular coupling response has been defined for the adult brain but in the neonate non‐invasive measurement of local cerebral perfusion using NIRS or BOLD fMRI have yielded variable and inconsistent results, including negative responses suggesting decreased perfusion and localised tissue tissue hypoxia. Also, the impact of permissive hypercapnia (PaCO2 > 50 mmHg) in the management of neonates on cerebrovascular responses to somatosensory input is unknown. Using NIRS to measure changes in cerebral oxy‐ and deoxy‐haemoglobin (ΔoxyHb, ΔdeoxyHb) in 8 anaesthetised newborn lambs, we studied the cerebral haemodynamic functional response to left median nerve stimulation using stimulus trains of 1.8, 4.8 and 7.8 s. Stimulation always produced a somatosensory evoked response, and superficial cortical perfusion measured by Laser Doppler Flowmetry predominantly increased following median nerve stimulation. However, with 1.8 s stimulation, oxyHb responses in the contralateral hemisphere were either positive (i.e. increased oxyHb), negative, or absent; and with 4.8 and 7.8 s stimulations, both positive and negative responses were observed. Hypercapnia increased baseline oxyHb and total Hb consistent with cerebral vasodilatation, and 6 of 7 lambs tested showed increased Δtotal Hb responses after the 7.8 s stimulation; among which 4 lambs also showed increased ΔoxyHb responses. In 2 of 3 lambs, the negative ΔoxyHb response became a positive pattern during hypercapnia. These results show that instead of functional hyperaemia, somatosensory stimulation can evoke negative (decreased oxyHb, total Hb) functional responses in the neonatal brain suggestive of decreased local perfusion and vasoconstriction, and that hypercapnia produces both baseline hyperperfusion and increased functional hyperaemia.
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In humans, gait adapts to prolonged walking on a split‐belt treadmill, where one leg steps faster than the other, by gradually restoring the symmetry of interlimb kinematic variables, such as double support periods and step lengths, and by reducing muscle activity (EMG, electromyography). The adaptation is also characterized by reversing the asymmetry of interlimb variables observed during the early split‐belt period when returning to tied‐belt locomotion, termed an after‐effect. To determine if cats adapt to prolonged split‐belt locomotion and to assess if spinal locomotor circuits participate in the adaptation, we measured interlimb variables and EMG in intact and spinal‐transected cats before, during and after 10 min of split‐belt locomotion. In spinal cats only the hindlimbs performed stepping with the forelimbs stationary. In intact and spinal cats, step lengths and double support periods were, on average, symmetric, during tied‐belt locomotion. They became asymmetric during split‐belt locomotion and remained asymmetric throughout the split‐belt period. Upon returning to tied‐belt locomotion, symmetry was immediately restored. In intact cats, the mean EMG amplitude of hindlimb extensors increased during split‐belt locomotion and remained increased throughout the split‐belt period, whereas in spinal cats, EMG amplitude did not change. Therefore, the results indicate that the locomotor pattern of cats does not adapt to prolonged split‐belt locomotion, suggesting an important physiological difference in the control of locomotion between cats and humans. We propose that restoring left‐right symmetry is not required to maintain balance during prolonged asymmetric locomotion in the cat, a quadruped, as opposed to human bipedal locomotion.
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A dominant feature of the olfactory bulb response to odour is fast synchronized oscillations at beta (15–40 Hz) or gamma (40–90 Hz) frequencies, thought to be involved in integration of olfactory signals. Mechanistically, the bulb presents an interesting case study for understanding how beta/gamma oscillations arise. Fast oscillatory synchrony in the activity of output mitral cells (MCs) appears to result from interactions with GABAergic granule cells (GCs), yet the incidence of MC‐GC connections is very low, around 4%. Here, we combined computational and experimental approaches to examine how oscillatory synchrony can nevertheless arise, focusing mainly on activity between “non‐sister” MCs affiliated with different glomeruli (interglomerular synchrony). In a sparsely connected model of MCs and GCs, we found first that interglomerular synchrony was generally quite low, but could be increased by a factor of 4 by physiological‐levels of gap junctional coupling between sister MCs at the same glomerulus. This effect was due to enhanced mutually synchronizing interactions between MC and GC populations. The potent role of gap junctions was confirmed in patch‐clamp recordings in bulb slices from wild‐type and connexin 36‐knockout (KO) mice. KO reduced both beta/gamma local field potential oscillations as well as synchrony of inhibitory signals in pairs of non‐sister MCs. These effects were independent of potential KO‐actions on network excitation. Divergent synaptic connections did not contribute directly to the vast majority of synchronized signals. Thus, in a sparsely connected network, gap junctions between a small subset of cells can, through population‐effects, greatly amplify oscillatory synchrony amongst unconnected cells.
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Key points
High work of breathing and exercise‐induced arterial hypoxaemia (EIAH) can decrease O2 delivery and exacerbate exercise‐induced quadriceps fatigue in healthy men.
Women have a higher work of breathing during exercise, dedicate a greater fraction of whole‐body V̇O2 towards their respiratory muscles and develop EIAH.
Despite a greater reduction in men's work of breathing, the attenuation of quadriceps fatigue was similar between the sexes.
The degree of EIAH was similar between sexes, and regardless of sex, those who developed the greatest hypoxaemia during exercise demonstrated the most attenuation of quadriceps fatigue.
Based on our previous finding that women have a greater relative oxygen cost of breathing, women appear to be especially susceptible to work of breathing‐related changes in quadriceps muscle fatigue.
Abstract
Reducing the work of breathing or eliminating exercise‐induced arterial hypoxaemia (EIAH) during exercise decreases the severity of quadriceps fatigue in men. Women have a greater work of breathing during exercise, dedicate a greater fraction of whole‐body V̇O2 towards their respiratory muscles, and demonstrate EIAH, suggesting women may be especially susceptible to quadriceps fatigue. Healthy subjects (8 male, 8 female) completed three constant load exercise tests over 4 days. During the first (control) test, subjects exercised at ∼85% of maximum while arterial blood gases and work of breathing were assessed. Subsequent constant load exercise tests were iso‐time and iso‐work rate, but with EIAH prevented by inspiring hyperoxic gas or work of breathing reduced via a proportional assist ventilator (PAV). Quadriceps fatigue was assessed by measuring force in response to femoral nerve stimulation. For both sexes, quadriceps force was equally reduced after the control trial (−27 ± 2% baseline) and was attenuated with hyperoxia and PAV (−18 ± 1 and −17 ± 2% baseline, P

Aberrant intracellular Ca2+ signalling contributes to the formation and progression of a range of distinct pathologies including cancers. Rises in intracellular Ca2+ concentration occur in response to Ca2+ influx through plasma membrane channels and Ca2+ release from intracellular Ca2+ stores, which can be mobilised in response to activation of cell surface receptors. OGR1 (Ovarian cancer G protein coupled Receptor 1, aka GPR68) is a proton‐sensing Gq‐coupled receptor that is most highly expressed in cerebellum. Medulloblastoma (MB) is the most common paediatric brain tumour that arises from cerebellar precursor cells. We find that nine distinct human MB samples all express OGR1. In both normal granule cells and the transformed human cerebellar granule cell line DAOY, OGR1 promotes expression of the proton‐potentiated member of the Canonical Transient Receptor Potential (TRPC) channel family, TRPC4. Consistent with a role for TRPC4 in MB, we find that all MB samples also express TRPC4. In DAOY cells, activation of TRPC4‐containing channels resulted in large Ca2+ influx and enhanced migration, while in normal cerebellar granule (precursor) cells and MB cells not derived from granule precursors, only small levels of Ca2+ influx and no enhanced migration was observed. Our results suggest that OGR1‐dependent increases in TRPC4 expression may favour formation of highly Ca2+‐permeable TRPC4‐containing channels that promote transformed granule cell migration. Increased motility of cancer cells is a prerequisite for cancer invasion and metastasis, and our findings may point towards a key role for TRPC4 in progression of certain types of MB.
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Key points
Central hypercapnic hypoventilation is highly prevalent in children suffering from congenital central hypoventilation syndrome (CCHS).
Mutations of the gene for paired‐like homeobox 2b (Phox2b) are aetiologically associated with CCHS and Phox2b is present in central components of respiratory chemoreflex, such as the nucleus tractus solitarius (NTS).
Injection of the neurotoxin substance P‐saporin into NTS destroys Phox2b‐expressing neurons. Impaired hypercapnic ventilatory response caused by this neurotoxin is attributable to a loss of CO2‐sensitive Phox2b‐expressing NTS neurons.
A subgroup of Phox2b‐expressing neurons exhibits intrinsic chemosensitivity. A background K+ channel‐like current is partially responsible for such chemosensitivity in Phox2b‐expressing neurons.
The present study helps us better understand the mechanism of respiratory deficits in CCHS and potentially locates a brainstem site for development of precise clinical intervention.
Abstract
The nucleus tractus solitarius (NTS) neurons have been considered to function as central respiratory chemoreceptors. However, the common molecular marker defined for these neurons remains unknown. The present study investigated whether paired‐like homeobox 2b (Phox2b)‐expressing NTS neurons are recruited in hypercapnic ventilatory response (HCVR) and whether these neurons exhibit intrinsic chemosensitivity. HCVR was assessed using whole body plethysmography and neuronal chemosensitivity was examined by patch clamp recordings in brainstem slices or dissociated neurons from Phox2b‐EGFP transgenic mice. Injection of the neurotoxin substance P‐saporin (SSP‐SAP) into NTS destroyed Phox2b‐expressing neurons. Minute ventilation and tidal volume were both reduced by 13% during exposure to 8% CO2 in inspired air when ∼13% of the Phox2b‐expressing neurons were eliminated. However, a loss of ∼18% of these neurons was associated with considerable decreases in minute ventilation by ≥18% and in tidal volume by≥22% when challenged by ≥4% CO2. In both cases, breathing frequency was unaffected. Most CO2‐activated neurons were immunoreactive to Phox2b. In brainstem slices, ∼43% of Phox2b‐expressing neurons from Phox2b‐EGFP mice displayed a sustained or transient increase in firing rate during physiological acidification (pH 7.0 or 8% CO2). Such a response was also present in dissociated neurons in favour of an intrinsic property. In voltage clamp recordings, a background K+ channel‐like current was found in a subgroup of Phox2b‐expressing neurons. Thus, the respiratory deficits caused by injection of SSP‐SAP into the NTS are attributable to proportional lesions of CO2/H+‐sensitive Phox2b‐expressing neurons.

Key points
Extracellular ATP, in association with [Ca2+]i regulation, is required to maintain basal ciliary beat frequency.
Increasing extracellular ATP levels increases ciliary beating in airway epithelial cells, maintaining a sustained response by inducing the release of additional ATP.
Extracellular ATP levels in the millimolar range, previously associated with pathophysiological conditions of the airway epithelium, produce a transient arrest of ciliary activity.
The regulation of ciliary beat frequency is dependent on ATP release by hemichannels (connexin/pannexin) and P2X receptor activation, the blockage of which may even stop ciliary movement.
The force exerted by cilia, measured by atomic force microscopy, is reduced following extracellular ATP hydrolysis. This result complements the current understanding of the ciliary beating regulatory mechanism, with special relevance to inflammatory diseases of the airway epithelium that affect mucociliary clearance.
Abstract
Extracellular nucleotides, including ATP, are locally released by the airway epithelium and stimulate ciliary activity in a [Ca2+]i‐dependent manner after mechanical stimulation of ciliated cells. However, it is unclear whether the ATP released is involved in regulating basal ciliary activity and mediating changes in ciliary activity in response to chemical stimulation. In the present study, we evaluated ciliary beat frequency (CBF) and ciliary beating forces in primary cultures from mouse tracheal epithelium, using videomicroscopy and atomic force microscopy (AFM), respectively. Extracellular ATP levels and [Ca2+]i were measured by luminometric and fluorimetric assays, respectively. Uptake of ethidium bromide was measured to evaluate hemichannel functionality. We show that hydrolysis of constitutive extracellular ATP levels with apyrase (50 U ml−1) reduced basal CBF by 45% and ciliary force by 67%. The apyrase effect on CBF was potentiated by carbenoxolone, a hemichannel inhibitor, and oxidized ATP, an antagonist used to block P2X7 receptors, which reduced basal CBF by 85%. Additionally, increasing extracellular ATP levels (0.1–100 μm) increased CBF, maintaining a sustained response that was suppressed in the presence of carbenoxolone. We also show that high levels of ATP (1 mm), associated with inflammatory conditions, lowered basal CBF by reducing [Ca2+]i and hemichannel functionality. In summary, we provide evidence indicating that airway epithelium ATP release is the molecular autocrine mechanism regulating basal ciliary activity and is also the mediator of the ciliary response to chemical stimulation.

[Ca2+]i must be low enough in diastole so that the ventricle is relaxed and can refill with blood. Interference with this will impair relaxation. The factors responsible for regulation of diastolic [Ca2+]i, in particular the relative roles of the sarcoplasmic reticulum (SR) and surface membrane are unclear. We investigated the effects on diastolic [Ca2+]i that result from the changes of Ca cycling known to occur in heart failure. Experiments were performed using Fluo‐3 in voltage‐clamped rat ventricular myocytes. Increasing stimulation frequency increased diastolic [Ca2+]i. This increase of [Ca2+]i was larger when SR function was impaired either by making the RyR leaky (with caffeine or ryanodine) or by decreasing SERCA activity with thapsigargin. The increase of diastolic [Ca2+]i produced by interfering with the SR was accompanied by a decrease of the amplitude of the systolic Ca transient such that there was no change of time‐averaged [Ca2+]i. Time‐averaged [Ca2+]i was increased by β‐adrenergic stimulation with isoprenaline and increased in a saturating manner with increased stimulation frequency; average [Ca2+]i was a linear function of Ca entry per unit time. Diastolic and time‐averaged [Ca2+]i were decreased by decreasing the L‐type Ca current (with 50 μm cadmium chloride). We conclude that diastolic [Ca2+]i is controlled by the balance between Ca entry and efflux during systole. Furthermore, manoeuvres which decrease the amplitude of the Ca transient (without decreasing Ca influx) will therefore increase diastolic [Ca2+]i. This identifies a novel mechanism whereby changes of the amplitude of the systolic Ca transient control diastolic [Ca2+]i.
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It is commonly agreed that the main function of the cerebellar system is to provide well‐timed signals used for the execution of motor commands or prediction of sensory inputs. This function is manifested as a temporal sequence of spiking that should be expressed in the cerebellar nuclei projection neurons (CN). Whether spiking activity is generated by excitation or release from inhibition is still a hotly debated issue.
In an attempt to resolve this debate, we recorded intracellularly from CN neurons in anaesthetized mice and performed an analysis of synaptic activity that yielded a number of important observations. First, we demonstrate that CN neurons can be classified into four groups. Second, shape‐index plots of the excitatory events suggest that they are distributed all over the dendritic tree. Third, the rise time of excitatory events is linearly related to the amplitude, suggesting that all excitatory events contribute equally to the generation of action potentials. Fourth, we identified a temporal pattern of spontaneous excitatory events that represent climbing fibre inputs and confirm the results by direct stimulation and analysis on harmaline evoked activity. Finally, we demonstrate that the probability of excitatory inputs to generate an AP is 0.1 yet half of the APs are generated by excitatory events. Moreover, the probability of a presumably spontaneous climbing fibre input to generate an AP is higher, reaching mean population value of 0.15. In view of these results, the mode of synaptic integration at the level of the CN should be re‐considered.
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Endothelial mechanotransduction is important for vascular function but alterations and activation of vascular mechanosensory proteins have not been investigated in humans. In endothelial cell culture, simple sugars effectively impair mechanosensor proteins. To study mechanosensor‐ and vascular function in humans, twelve young healthy male subjects supplemented their diet with 3 × 75 g sucrose day−1 for 14 days in a randomized cross‐over design. Before and after the intervention period, the hyperemic response to passive lower leg movement and active knee extensor exercise was determined by ultrasound doppler. A muscle biopsy was obtained from the thigh muscle before and after acute passive leg movement, to asses the protein amount and phosphorylation status of mechanosensory proteins and NADPH oxidase. The sucrose intervention led to a reduced flow response to passive movement (by 17 ± 2 %) and to 12 watts of active exercise (by 9 ± 1 %), indicating impaired vascular function. Reduced flow response to passive and active exercise was paralleled by a significant upregulation of Platelet endothelial cell adhesion molecule (PECAM‐1), endothelial nitric oxide synthase, NADPH oxidase and the Rho family GTPase Rac1 protein expression in the muscle tissue as well as an increased basal phosphorylation status of Vascular endothelial growth factor receptor 2 and a reduced phosphorylation status of PECAM‐1. The phosphorylation status was not acutely altered with passive leg movement. These findings indicate that regular intake of high levels of sucrose can impair vascular mechanotransduction, increase the oxidative stress potential and suggest that dietary excessive sugar intake may contribute to the development of vascular disease.
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Voltage‐gated sodium channels are critical for neuronal activity, and highly intolerant to variation. Even mutations that cause subtle changes in the activity these channels are sufficient to cause devastating inherited neurological diseases, such as epilepsy and pain. However, these channels do vary in healthy tissue. Alternative splicing modifies sodium channels, but the functional relevance and adaptive significance of this splicing remain poorly understood. Here we use a conserved alternate exon encoding part of the first domain of sodium channels to compare how splicing modifies different channels, and to ask whether the functional consequences of this splicing have been preserved in different genes. Although the splicing event is highly conserved, one splice variant has been selectively removed from Nav1.1 in multiple mammalian species, suggesting that the functional variation in Nav1.1 is less well‐tolerated. We show for three human channels (Nav1.1, Nav1.2 and Nav1.7) splicing modifies the return from inactivated to deactivated states, and the differences between splice variants are occluded by antiepileptic drugs that bind to and stabilize inactivated states. A model based on structural data can replicate these changes, and indicates that splicing may exploit a distinct role of the first domain to change channel availability, and that the first domain of all three sodium channels plays a role in determining the rate at which the inactivation domain dissociates. Taken together, our data suggest that the stability of inactivated states is under tight evolutionary control, but that in Nav1.1 faster recovery from inactivation is associated with negative selection in mammals.
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Key points
This study represents a first step toward predicting mechanisms of sex‐based arrhythmias that may lead to important developments in risk stratification and may inform future drug design and screening.
We undertook simulations to reveal the conditions (i.e. pacing, drugs, sympathetic stimulation) required for triggering and sustaining reentrant arrhythmias.
Using the recently solved cryo‐EM structure for the Eag‐family channel as a template, we revealed potential interactions of oestrogen with the pore loop hERG mutation (G604S).
Molecular models suggest that oestrogen and dofetilide blockade can concur simultaneously in the hERG channel pore.
Abstract
Female sex is a risk factor for inherited and acquired long‐QT associated torsade de pointes (TdP) arrhythmias, and sympathetic discharge is a major factor in triggering TdP in female long‐QT syndrome patients. We used a combined experimental and computational approach to predict ‘the perfect storm’ of hormone concentration, IKr block and sympathetic stimulation that induces arrhythmia in females with inherited and acquired long‐QT. More specifically, we developed mathematical models of acquired and inherited long‐QT syndrome in male and female ventricular human myocytes by combining effects of a hormone and a hERG blocker, dofetilide, or hERG mutations. These ‘male’ and ‘female’ model myocytes and tissues then were used to predict how various sex‐based differences underlie arrhythmia risk in the setting of acute sympathetic nervous system discharge. The model predicted increased risk for arrhythmia in females when acute sympathetic nervous system discharge was applied in the settings of both inherited and acquired long‐QT syndrome. Females were predicted to have protection from arrhythmia induction when progesterone is high. Males were protected by the presence of testosterone. Structural modelling points towards two plausible and distinct mechanisms of oestrogen action enhancing torsadogenic effects: oestradiol interaction with hERG mutations in the pore loop containing G604 or with common TdP‐related blockers in the intra‐cavity binding site. Our study presents findings that constitute the first evidence linking structure to function mechanisms underlying female dominance of arousal‐induced arrhythmias.

Key points
Patients with post‐traumatic stress disorder (PTSD) are at a significantly higher risk of developing hypertension and cardiovascular disease. The mechanisms underlying this increased risk are not known.
Studies have suggested that PTSD patients have an overactive sympathetic nervous system (SNS) that could contribute to cardiovascular risk; however, sympathetic function has not previously been rigorously evaluated in PTSD patients.
Using direct measurements of sympathetic nerve activity and pharmacological manipulation of blood pressure, we show that veterans with PTSD have augmented SNS and haemodynamic reactivity during both combat‐related and non‐combat related mental stress, impaired sympathetic and cardiovagal baroreflex sensitivity, and increased inflammation.
Identifying the mechanisms contributing to increased cardiovascular (CV) risk in PTSD will pave the way for developing interventions to improve sympathetic function and reduce CV risk in these patients.
Abstract
Post‐traumatic stress disorder (PTSD) is associated with increased cardiovascular (CV) risk. We tested the hypothesis that PTSD patients have augmented sympathetic nervous system (SNS) and haemodynamic reactivity during mental stress, as well as impaired arterial baroreflex sensitivity (BRS). Fourteen otherwise healthy Veterans with combat‐related PTSD were compared with 14 matched Controls without PTSD. Muscle sympathetic nerve activity (MSNA), continuous blood pressure (BP) and electrocardiography were measured at baseline, as well as during two types of mental stress: combat‐related mental stress using virtual reality combat exposure (VRCE) and non‐combat related stress using mental arithmetic (MA). A cold pressor test (CPT) was administered for comparison. BRS was tested using pharmacological manipulation of BP via the Modified Oxford technique at rest and during VRCE. Blood samples were analysed for inflammatory biomarkers. Baseline characteristics, MSNA and haemodynamics were similar between the groups. In PTSD vs. Controls, MSNA (+8.2 ± 1.0 vs. +1.2 ± 1.3 bursts min–1, P

Key points
Hydrocortisone (HC) is required for activation of large‐conductance Ca2+‐activated K+ current (BK) by purinergic receptor agonists.
HC reduces insertion of the stress‐regulated exon (STREX) in the KCNMA1 gene, permitting protein kinase C (PKC)‐dependent channel activation.
Overlapping and unique purinergic signalling regions exist at the apical border of differentiated surface cells.
BK channels localize in the cilia of surface cells.
Abstract
In the present study we investigated the role of hydrocortisone (HC) on uridine‐5ʹ‐triphosphate (UTP)‐stimulated ion transport in differentiated, pseudostratified epithelia derived from normal human bronchial basal cells. The presence of a UTP‐stimulated, paxilline‐sensitive large‐conductance Ca2+‐activated K+ (BK) current was demonstrated in control epithelia but was not stimulated in epithelia differentiated in the absence of HC (HC0). Addition of the BK channel opener NS11021 directly activated channels in control epithelia; however, under HC0 conditions, activation only occurred when UTP was added after NS11021. The PKC inhibitors GF109203x and Gö6983 blocked BK activation by UTP in control epithelia, suggesting that PKC‐mediated phosphorylation plays a permissive role in purinoceptor‐stimulated BK activation. Moreover, HC0 epithelia expressed significantly more KCNMA1 containing the stress‐regulated exon (STREX), a splice‐variant of the α‐subunit that displays altered channel regulation by phosphorylation, compared to control epithelia. Furthermore, BK channels as well as purinergic receptors were shown to localize in unique and overlapping domains at the apical membrane of ciliated surface cells. These results establish a previously unrecognized role for glucocorticoids in regulation of BK channels in airway epithelial cells.

Key points
Exercise is a potent physiological stimulus to clear blood glucose from the circulation into skeletal muscle.
The mammalian target of rapamycin complex 2 (mTORC2) is an important regulator of muscle glucose uptake in response to insulin stimulation.
Here we report for the first time that the activity of mTORC2 in mouse muscle increases during exercise.
We further show that glucose uptake during exercise is decreased in mouse muscle that lacks mTORC2 activity.
We also provide novel identifications of new mTORC2 substrates during exercise in mouse muscle.
Abstract
Exercise increases glucose uptake into insulin‐resistant muscle. Thus, elucidating the exercise signalling network in muscle may uncover new therapeutic targets. The mammalian target of rapamycin complex 2 (mTORC2), a regulator of insulin‐controlled glucose uptake, has been reported to interact with ras‐related C3 botulinum toxin substrate 1 (Rac1), which plays a role in exercise‐induced glucose uptake in muscle. Therefore, we tested the hypothesis that mTORC2 activity is necessary for muscle glucose uptake during treadmill exercise. We used mice that specifically lack mTORC2 signalling in muscle by deletion of the obligatory mTORC2 component Rictor (Ric mKO). Running capacity and running‐induced changes in blood glucose, plasma lactate and muscle glycogen levels were similar in wild‐type (Ric WT) and Ric mKO mice. At rest, muscle glucose uptake was normal, but during running muscle glucose uptake was reduced by 40% in Ric mKO mice compared to Ric WT mice. Running increased muscle phosphorylated 5′ AMP‐activated protein kinase (AMPK) similarly in Ric WT and Ric mKO mice, and glucose transporter type 4 (GLUT4) and hexokinase II (HKII) protein expressions were also normal in Ric mKO muscle. The mTORC2 substrate, phosphorylated protein kinase C α (PKCα), and the mTORC2 activity readout, phosphorylated N‐myc downstream regulated 1 (NDRG1) protein increased with running in Ric WT mice, but were not altered by running in Ric mKO muscle. Quantitative phosphoproteomics uncovered several additional potential exercise‐dependent mTORC2 substrates, including contractile proteins, kinases, transcriptional regulators, actin cytoskeleton regulators and ion‐transport proteins. Our study suggests that mTORC2 is a component of the exercise signalling network that regulates muscle glucose uptake and we provide a resource of new potential members of the mTORC2 signalling network.

Key points
Mitochondrial function changes over time at high altitudes, but the potential benefits of these changes for hypoxia resistance remains unclear.
We used high‐altitude‐adapted populations of deer mice, which exhibit enhanced aerobic performance in hypoxia, to examine whether changes in mitochondrial physiology or intracellular distribution in the muscle contribute to hypoxia resistance.
Permeabilized muscle fibres from the gastrocnemius muscle had higher respiratory capacities in high‐altitude mice than in low‐altitude mice.
Highlanders also had higher mitochondrial volume densities, due entirely to an enriched abundance of subsarcolemmal mitochondria, such that more mitochondria were situated near the cell membrane and adjacent to capillaries.
There were several effects of hypoxia acclimation on mitochondrial function, some of which were population specific, but they differed from the evolved changes in high‐altitude natives, which probably provide a better indication of adaptive traits that improve performance and hypoxia resistance at high altitudes.
Abstract
High‐altitude natives that have evolved to live in hypoxic environments provide a compelling system to understand how animals can overcome impairments in oxygen availability. We examined whether these include changes in mitochondrial physiology or intracellular distribution that contribute to hypoxia resistance in high‐altitude deer mice (Peromyscus maniculatus). Mice from populations native to high and low altitudes were born and raised in captivity, and as adults were acclimated to normoxia or hypobaric hypoxia (equivalent to 4300 m elevation). We found that highlanders had higher respiratory capacities in the gastrocnemius (but not soleus) muscle than lowlanders (assessed using permeabilized fibres with single or multiple inputs to the electron transport system), due in large part to higher mitochondrial volume densities in the gastrocnemius. The latter was attributed to an increased abundance of subsarcolemmal (but not intermyofibrillar) mitochondria, such that more mitochondria were situated near the cell membrane and adjacent to capillaries. Hypoxia acclimation had no significant effect on these population differences, but it did increase mitochondrial cristae surface densities of mitochondria in both populations. Hypoxia acclimation also altered the physiology of isolated mitochondria by affecting respiratory capacities and cytochrome c oxidase activities in population‐specific manners. Chronic hypoxia decreased the release of reactive oxygen species by isolated mitochondria in both populations. There were subtle differences in O2 kinetics between populations, with highlanders exhibiting increased mitochondrial O2 affinity or catalytic efficiency in some conditions. Our results suggest that evolved changes in mitochondrial physiology in high‐altitude natives are distinct from the effects of hypoxia acclimation, and probably provide a better indication of adaptive traits that improve performance and hypoxia resistance at high altitudes.

Key points
Loss of muscle mass and strength in the growing population of elderly people is a major health concern for modern societies. This condition, termed sarcopenia, is a major cause of falls and of the subsequent increase in morbidity and mortality.
Despite numerous studies on the impact of ageing on individual muscle fibres, the contribution of single muscle fibre adaptations to ageing‐induced atrophy and functional impairment is still unsettled.
The level of physical function and disuse is often associated with ageing.
We studied relatively healthy older adults in order to understand the effects of ageing per se without the confounding impact of impaired physical function.
We found that in healthy ageing, structural and functional alterations of muscle fibres occur. Protein post‐translational modifications, oxidation and phosphorylation contribute to such alterations more than loss of myosin and other muscle protein content.
Abstract
Contradictory results have been reported on the impact of ageing on structure and functions of skeletal muscle fibres, likely to be due to a complex interplay between ageing and other phenomena such as disuse and diseases. Here we recruited healthy, physically and socially active young (YO) and elderly (EL) men in order to study ageing per se without the confounding effects of impaired physical function. In vivo analyses of quadriceps and in vitro analyses of vastus lateralis muscle biopsies were performed. In EL subjects, our results show that (i) quadriceps volume, maximum voluntary contraction isometric torque and patellar tendon force were significantly lower; (ii) muscle fibres went through significant atrophy and impairment of specific force (isometric force/cross‐sectional area) and unloaded shortening velocity; (iii) myosin/actin ratio and myosin content in individual muscle fibres were not altered; (iv) the muscle proteome went through quantitative adaptations, namely an up‐regulation of the content of several groups of proteins among which were myofibrillar proteins and antioxidant defence systems; (v) the muscle proteome went through qualitative adaptations, namely phosphorylation of several proteins, including myosin light chain‐2 slow and troponin T and carbonylation of myosin heavy chains. The present results indicate that impairment of individual muscle fibre structure and function is a major feature of ageing per se and that qualitative adaptations of muscle proteome are likely to be more involved than quantitative adaptations in determining such a phenomenon.

Key points
The catechol metabolites of 17β‐oestradiol (E2β), 2‐hydroxyoestradiol (2‐OHE2) and 4‐hydroxyoestradiol (4‐OHE2), stimulate proliferation of pregnancy‐derived ovine uterine artery endothelial cells (P‐UAECs) through β‐adrenoceptors (β‐ARs) and independently of the classic oestrogen receptors (ERs).
Herein we show that activation of ERK1/2, p38 and JNK mitogen activated protein kinases (MAPKs) is necessary for 2‐OHE2‐ and 4‐OHE2‐induced P‐UAEC proliferation, as well as proliferation induced by the parent hormone E2β and other β‐AR signalling hormones (i.e. catecholamines).
Conversely, although 2‐OHE2 and 4‐OHE2 rapidly activate phosphatidylinositol 3‐kinase (PI3K), its activation is not involved in catecholoestradiol‐induced P‐UAEC proliferation.
We also show for the first time the signalling mechanisms involved in catecholoestradiol‐induced P‐UAEC proliferation; which converge at the level of MAPKs with the signalling mechanisms mediating E2β‐ and catecholamine‐induced proliferation.
The present study advances our understanding of the complex signalling mechanisms involved in regulating uterine endothelial cell proliferation during pregnancy.
Abstract
Previously we demonstrated that the biologically active metabolites of 17β‐oestradiol, 2‐hydroxyoestradiol (2‐OHE2) and 4‐hydroxyoestradiol (4‐OHE2), stimulate pregnancy‐specific proliferation of uterine artery endothelial cells derived from pregnant (P‐UAECs), but not non‐pregnant ewes. However, unlike 17β‐oestradiol, which induces proliferation via oestrogen receptor‐β (ER‐β), the catecholoestradiols mediate P‐UAEC proliferation via β‐adrenoceptors (β‐AR) and independently of classic oestrogen receptors. Herein, we aim to further elucidate the signalling mechanisms involved in proliferation induced by catecholoestradiols in P‐UAECs. P‐UAECs were treated with 2‐OHE2 and 4‐OHE2 for 0, 0.25, 0.5, 1, 2, 4, 12 and 24 h, to analyse activation of mitogen activated protein kinases (MAPKs) and phosphatidylinositol 3‐kinase (PI3K)–AKT. Specific inhibitors for ERK1/2 MAPK (PD98059), p38 MAPK (SB203580), JNK MAPK (SP600125), or PI3K (LY294002) were used to determine the involvement of individual kinases in agonist‐induced P‐UAEC proliferation. 2‐OHE2 and 4‐OHE2 stimulated biphasic phosphorylation of ERK1/2, slow p38 and JNK phosphorylation over time, and rapid monophasic AKT phosphorylation. Furthermore, ERK1/2, p38 and JNK MAPKs, but not PI3K, were individually necessary for catecholoestradiol‐induced proliferation. In addition, when comparing the signalling mechanisms of the catecholoestradiols, to 17β‐oestradiol and catecholamines, we observed that convergent MAPKs signalling pathways facilitate P‐UAEC proliferation induced by all of these hormones. Thus, all three members of the MAPK family mediate the mitogenic effects of catecholoestradiols in the endothelium during pregnancy. Furthermore, the convergent signalling of MAPKs involved in catecholoestradiol‐, 17β‐oestradiol‐ and catecholamine‐induced endothelial cell proliferation may be indicative of unappreciated evolutionary functional redundancy to facilitate angiogenesis and ensure maintenance of uterine blood flow during pregnancy.

Key points
The angiotensin II receptor type 1b (AT1Rb) is the primary sensor of intraluminal pressure in cerebral arteries.
Pressure or membrane‐stretch induced stimulation of AT1Rb activates the TRPM4 channel and results in inward transient cation currents that depolarize smooth muscle cells, leading to vasoconstriction.
Activation of either AT1Ra or AT1Rb with angiotensin II stimulates TRPM4 currents in cerebral artery myocytes and vasoconstriction of cerebral arteries.
The expression of AT1Rb mRNA is ∼30‐fold higher than AT1Ra in whole cerebral arteries and ∼45‐fold higher in isolated cerebral artery smooth muscle cells.
Higher levels of expression are likely to account for the obligatory role of AT1Rb for pressure‐induced vasoconstriction.
Abstract
Myogenic vasoconstriction, which reflects the intrinsic ability of smooth muscle cells to contract in response to increases in intraluminal pressure, is critically important for the autoregulation of blood flow. In smooth muscle cells from cerebral arteries, increasing intraluminal pressure engages a signalling cascade that stimulates cation influx through transient receptor potential (TRP) melastatin 4 (TRPM4) channels to cause membrane depolarization and vasoconstriction. Substantial evidence indicates that the angiotensin II receptor type 1 (AT1R) is inherently mechanosensitive and initiates this signalling pathway. Rodents express two types of AT1R – AT1Ra and AT1Rb – and conflicting studies provide support for either isoform as the primary sensor of intraluminal pressure in peripheral arteries. We hypothesized that mechanical activation of AT1Ra increases TRPM4 currents to induce myogenic constriction of cerebral arteries. However, we found that development of myogenic tone was greater in arteries from AT1Ra knockout animals compared with controls. In patch‐clamp experiments using native cerebral arterial myocytes, membrane stretch‐induced cation currents were blocked by the TRPM4 inhibitor 9‐phenanthrol in both groups. Further, the AT1R blocker losartan (1 μm) diminished myogenic tone and blocked stretch‐induced cation currents in cerebral arteries from both groups. Activation of AT1R with angiotensin II (30 nm) also increased TRPM4 currents in smooth muscle cells and constricted cerebral arteries from both groups. Expression of AT1Rb mRNA was ∼30‐fold greater than AT1Ra in cerebral arteries, and knockdown of AT1Rb selectively diminished myogenic constriction. We conclude that AT1Rb, acting upstream of TRPM4 channels, is the primary sensor of intraluminal pressure in cerebral artery smooth muscle cells.

Key points
The heat of activation of cardiac muscle reflects the metabolic cost of restoring ionic homeostasis following a contraction. The accuracy of its measurement depends critically on the abolition of crossbridge cycling.
We abolished crossbridge activity in isolated rat ventricular trabeculae by use of blebbistatin, an agent that selectively inhibits myosin II ATPase.
We found cardiac activation heat to be muscle length independent and to account for 15–20% of total heat production at body temperature.
We conclude that it can be accurately estimated at minimal muscle length.
Abstract
Activation heat arises from two sources during the contraction of striated muscle. It reflects the metabolic expenditure associated with Ca2+ pumping by the sarcoplasmic reticular Ca2+‐ATPase and Ca2+ translocation by the Na+/Ca2+ exchanger coupled to the Na+,K+‐ATPase. In cardiac preparations, investigators are constrained in estimating its magnitude by reducing muscle length to the point where macroscopic twitch force vanishes. But this experimental protocol has been criticised since, at zero force, the observed heat may be contaminated by residual crossbridge cycling activity. To eliminate this concern, the putative thermal contribution from crossbridge cycling activity must be abolished, at least at minimal muscle length. We achieved this using blebbistatin, a selective inhibitor of myosin II ATPase. Using a microcalorimeter, we measured the force production and heat output, as functions of muscle length, of isolated rat trabeculae from both ventricles contracting isometrically at 5 Hz and at 37°C. In the presence of blebbistatin (15 μmol l−1), active force was zero but heat output remained constant, at all muscle lengths. Activation heat measured in the presence of blebbistatin was not different from that estimated from the intercept of the heat–stress relation in its absence. We thus reached two conclusions. First, activation heat is independent of muscle length. Second, residual crossbridge heat is negligible at zero active force; hence, the intercept of the cardiac heat–force relation provides an estimate of activation heat uncontaminated by crossbridge cycling. Both results resolve long‐standing disputes in the literature.

Key points
Mutations in genes encoding cardiac troponin I (TNNI3) and cardiac troponin T (TNNT2) caused altered troponin protein stoichiometry in patients with dilated cardiomyopathy.
TNNI3p.98trunc resulted in haploinsufficiency, increased Ca2+‐sensitivity and reduced length‐dependent activation.
TNNT2p.K217del caused increased passive tension.
A mutation in the gene encoding Lamin A/C (LMNAp.R331Q) led to reduced maximal force development through secondary disease remodelling in patients suffering from dilated cardiomyopathy.
Our study shows that different gene mutations induce dilated cardiomyopathy via diverse cellular pathways.
Abstract
Dilated cardiomyopathy (DCM) can be caused by mutations in sarcomeric and non‐sarcomeric genes. In this study we defined the pathogenic effects of three DCM‐causing mutations: the sarcomeric mutations in genes encoding cardiac troponin I (TNNI3p.98truncation) and cardiac troponin T (TNNT2p.K217deletion; also known as the p.K210del) and the non‐sarcomeric gene mutation encoding lamin A/C (LMNAp.R331Q). We assessed sarcomeric protein expression and phosphorylation and contractile behaviour in single membrane‐permeabilized cardiomyocytes in human left ventricular heart tissue. Exchange with recombinant troponin complex was used to establish the direct pathogenic effects of the mutations in TNNI3 and TNNT2. The TNNI3p.98trunc and TNNT2p.K217del mutation showed reduced expression of troponin I to 39% and 51%, troponin T to 64% and 53%, and troponin C to 73% and 97% of controls, respectively, and altered stoichiometry between the three cardiac troponin subunits. The TNNI3p.98trunc showed pure haploinsufficiency, increased Ca2+‐sensitivity and impaired length‐dependent activation. The TNNT2p.K217del mutation showed a significant increase in passive tension that was not due to changes in titin isoform composition or phosphorylation. Exchange with wild‐type troponin complex corrected troponin protein levels to 83% of controls in the TNNI3p.98trunc sample. Moreover, upon exchange all functional deficits in the TNNI3p.98trunc and TNNT2p.K217del samples were normalized to control values confirming the pathogenic effects of the troponin mutations. The LMNAp.R331Q mutation resulted in reduced maximal force development due to disease remodelling. Our study shows that different gene mutations induce DCM via diverse cellular pathways.

Key Points
Lamprey are cyclostomes, a group of vertebrates that diverged from lines leading to jawed vertebrates (including mammals) in the late Cambrian, 500 million years ago. It may therefore be possible to infer properties of photoreceptors in early vertebrate progenitors by comparing lamprey to other vertebrates.
We show that lamprey rods and cones respond to light much like rods and cones in amphibians and mammals. They operate over a similar range of light intensities and adapt to backgrounds and bleaches nearly identically.
These correspondences are pervasive and detailed; they argue for the presence of rods and cones very early in the evolution of vertebrates with properties much like those of rods and cones in existing vertebrate species.
Abstract
The earliest vertebrates were agnathans – fish‐like organisms without jaws, which first appeared near the end of the Cambrian radiation. One group of agnathans became cyclostomes, which include lamprey and hagfish. Other agnathans gave rise to jawed vertebrates or gnathostomes, the group including all other existing vertebrate species. Because cyclostomes diverged from other vertebrates 500 million years ago, it may be possible to infer some of the properties of the retina of early vertebrate progenitors by comparing lamprey to other vertebrates. We have previously shown that rods and cones in lamprey respond to light much like photoreceptors in other vertebrates and have a similar sensitivity. We now show that these affinities are even closer. Both rods and cones adapt to background light and to bleaches in a manner almost identical to other vertebrate photoreceptors. The operating range in darkness is nearly the same in lamprey and in amphibian or mammalian rods and cones; moreover background light shifts response–intensity curves downward and to the right over a similar range of ambient intensities. Rods show increment saturation at about the same intensity as mammalian rods, and cones never saturate. Bleaches decrease sensitivity in part by loss of quantum catch and in part by opsin activation of transduction. These correspondences are so numerous and pervasive that they are unlikely to result from convergent evolution but argue instead that early vertebrate progenitors of both cyclostomes and mammals had photoreceptors much like our own.

Key points
Regionalization of the stretch reflex, i.e. the notion that the activation of 1a afferents from a muscle region influences only the activation of motor units in the same region, has been demonstrated previously in animals but not in humans.
Mechanical stretches applied to regions of vastus medialis as close as 10 mm apart resulted in recruitment of motor units localized topographically with respect to the location of the mechanical stretch.
Stretch reflexes are regionalized in the human vastus medialis.
The human spinal cord has the neuromuscular circuitry to preferentially activate motoneurones innervating muscle fibres located in different regions of the vastus medialis.
Abstract
The localization of motor unit territories provides an anatomical basis to suggest that the CNS may have more independence in motor unit recruitment and control strategies than what was previously thought. In this study, we investigated whether the human spinal cord has the neuromuscular circuitry to independently activate motor units located in different regions of the vastus medialis. Mechanical taps were applied to multiple locations in the vastus medialis (VM) in nine healthy individuals. Regional responses within the muscle were observed using a grid of 5 × 13 surface EMG electrodes. The EMG amplitude was quantified for each channel, and a cluster of channels showing the largest activation was identified. The spatial location of the EMG response was quantified as the position of the channels in the cluster. In a subset of three participants, intramuscular recordings were performed simultaneously with the surface EMG recordings. Mechanical taps resulted in localized, discrete responses for each participant. The spatial location of the elicited responses was dependent on the location of the tap (P

Key points
Small transmembrane proteins such as FXYDs, which interact with Na+,K+‐ATPase, and the micropeptides that interact with sarco/endoplasmic reticulum Ca2+‐ATPase play fundamental roles in regulation of ion transport in vertebrates.
Uncertain evolutionary origins and phylogenetic relationships among these regulators of ion transport have led to inconsistencies in their classification across vertebrate species, thus hampering comparative studies of their functions.
We discovered the first FXYD homologue in sea lamprey, a basal jawless vertebrate, which suggests small transmembrane regulators of ion transport emerged early in the vertebrate lineage.
We also identified 13 gene subfamilies of FXYDs and propose a revised, phylogeny‐based FXYD classification that is consistent across vertebrate species.
These findings provide an improved framework for investigating physiological and pathophysiological functions of small transmembrane regulators of ion transport.
Abstract
Small transmembrane proteins are important for regulation of cellular ion transport. The most prominent among these are members of the FXYD family (FXYD1–12), which regulate Na+,K+‐ATPase, and phospholamban, sarcolipin, myoregulin and DWORF, which regulate the sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA). FXYDs and regulators of SERCA are present in fishes, as well as terrestrial vertebrates; however, their evolutionary origins and phylogenetic relationships are obscure, thus hampering comparative physiological studies. Here we discovered that sea lamprey (Petromyzon marinus), a representative of extant jawless vertebrates (Cyclostomata), expresses an FXYD homologue, which strongly suggests that FXYDs predate the emergence of fishes and other jawed vertebrates (Gnathostomata). Using a combination of sequence‐based phylogenetic analysis and conservation of local chromosome context, we determined that FXYDs markedly diversified in the lineages leading to cartilaginous fishes (Chondrichthyes) and bony vertebrates (Euteleostomi). Diversification of SERCA regulators was much less extensive, indicating they operate under different evolutionary constraints. Finally, we found that FXYDs in extant vertebrates can be classified into 13 gene subfamilies, which do not always correspond to the established FXYD classification. We therefore propose a revised classification that is based on evolutionary history of FXYDs and that is consistent across vertebrate species. Collectively, our findings provide an improved framework for investigating the function of ion transport in health and disease.

Key points
Both endogenous opioids and opiate drugs of abuse modulate learning of habitual and goal‐directed actions, and can also modify long‐term plasticity of corticostriatal synapses.
Striatal projection neurons of the direct pathway co‐release the opioid neuropeptide dynorphin which can inhibit dopamine release via κ‐opioid receptors.
Theta‐burst stimulation of corticostriatal fibres produces long‐term potentiation (LTP) in striatal projection neurons when measured using whole‐cell patch recording.
Optogenetic activation of direct pathway striatal projection neurons inhibits LTP while reducing dopamine release.
Because the endogenous release of opioids is activity dependent, this modulation of synaptic plasticity represents a negative feedback mechanism that may limit runaway enhancement of striatal neuron activity in response to drugs of abuse.
Abstract
Synaptic plasticity in the striatum adjusts behaviour adaptively during skill learning, or maladaptively in the case of addiction. Just as dopamine plays a critical role in synaptic plasticity underlying normal skill learning and addiction, endogenous and exogenous opiates also modulate learning and addiction‐related striatal plasticity. Though the role of opioid receptors in long‐term depression in striatum has been characterized, their effect on long‐term potentiation (LTP) remains unknown. In particular, direct pathway (dopamine D1 receptor‐containing; D1R‐) spiny projection neurons (SPNs) co‐release the opioid neuropeptide dynorphin, which acts at presynaptic κ‐opioid receptors (KORs) on dopaminergic afferents and can negatively regulate dopamine release. Therefore, we evaluated the interaction of co‐released dynorphin and KOR on striatal LTP. We optogenetically facilitate the release of endogenous dynorphin from D1R‐SPNs in brain slice while using whole‐cell patch recording to measure changes in the synaptic response of SPNs following theta‐burst stimulation (TBS) of cortical afferents. Our results demonstrate that TBS evokes corticostriatal LTP, and that optogenetic activation of D1R‐SPNs during induction impairs LTP. Additional experiments demonstrate that optogenetic activation of D1R‐SPNs reduces stimulation‐evoked dopamine release and that bath application of a KOR antagonist provides full rescue of both LTP induction and dopamine release during optogenetic activation of D1R‐SPNs. These results suggest that an increase in the opioid neuropeptide dynorphin is responsible for reduced TBS LTP and illustrate a physiological phenomenon whereby heightened D1R‐SPN activity can regulate corticostriatal plasticity. Our findings have important implications for learning in addictive states marked by elevated direct pathway activation.

Key points
The mucosa may have neuron‐like functions as urinary bladder mucosa releases bioactive substances that modulate sensory nerve activity as well as detrusor muscle contractility. However, such mucosal function in other visceral organs remains to be established.
The role of mucosa in generating spontaneous contractions in seminal vesicles (SVs), a paired organ in the male reproductive tract, was investigated.
The intact mucosa is essential for the generation of spontaneous phasic contractions of SV smooth muscle arising from electrical slow waves and corresponding increases in intracellular Ca2+. These spontaneous events primarily depend on Ca2+ handling by sarco‐endoplasmic reticulum Ca2+ stores.
A population of mucosal cells developed spontaneous rises in intracellular Ca2+ relying on sarco‐endoplasmic reticulum Ca2+ handling.
The spontaneously active cells in the SV mucosa appear to drive spontaneous activity in smooth muscle either by sending depolarizing signals and/or by releasing humoral substances.
Abstract
The role of the mucosa in generating the spontaneous activity of guinea‐pig seminal vesicle (SV) was explored. Changes in contractility, membrane potential and intracellular Ca2+ dynamics of SV smooth muscle cells (SMCs) were recorded using isometric tension recording, intracellular microelectrode recording and epi‐fluorescence Ca2+ imaging, respectively. Mucosa‐intact but not mucosa‐denuded SV preparations generated TTX‐ (1 μm) resistant spontaneous phasic contractions that were abolished by nifedipine (3 μm). Consistently, SMCs developed mucosa‐dependent slow waves (SWs) that triggered action potentials and corresponding Ca2+ flashes. Nifedipine (10 μm) abolished the action potentials and spontaneous contractions, while suppressing the SWs and Ca2+ flashes. Both the residual SWs and spontaneous Ca2+ transients were abolished by cyclopiazonic acid (CPA, 10 μm), a sarco‐endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitor. DIDS (300 μm) and niflumic acid (100 μm), blockers for Ca2+‐activated Cl− channels (CACCs), or low Cl− solution also slowed or prevented the generation of SWs. In SV mucosal preparations detached from the muscle layer, a population of mucosal cells generated spontaneous Ca2+ transients that were blocked by CPA but not nifedipine. These results suggested that spontaneous contractions and corresponding Ca2+ flashes in SV SMCs arise from action potential generation due to the opening of L‐type voltage‐dependent Ca2+ channels. Spontaneous Ca2+ transients appear to primarily result from Ca2+ release from sarco‐endoplasmic reticulum Ca2+ stores to activate CACCs to develop SWs. The mucosal cells firing spontaneous Ca2+ transients may play a critical role in driving spontaneous activity of SV smooth muscle either by sending depolarizing signals or by releasing humoral substances.

Key points
α‐linolenic acid (ALA) and exercise training both attenuate hyperlipidaemia‐related cardiovascular derangements, however, there is a paucity of information pertaining to their mechanisms of action when combined.
We investigated both the independent and combined effects of exercise training and ALA consumption in obese Zucker rats, aiming to determine the potential for additive improvements in cardiovascular function.
ALA and exercise training independently improved cardiac output, end‐diastolic volume, left ventricular fibrosis and mean blood pressure following a 4 week intervention.
Combining ALA and endurance exercise yielded greater improvements in these parameters, independent of changes in markers of oxidative stress or endogenous anti‐oxidants.
We postulate that divergent mechanisms of action may explain these changes: ALA increases peripheral vasodilation, and exercise training stimulates angiogenesis.
Abstract
Although α‐linolenic acid (ALA) and endurance exercise training independently attenuate hyperlipidaemia‐related cardiovascular derangements, there is a paucity of information pertaining to their mechanisms of action and efficacy when combined as a preventative therapeutic approach. Therefore, we used obese Zucker rats to investigate the independent and combined effects of these interventions on cardiovascular disease. Specifically, animals were randomly assigned to one of the following groups: control diet‐sedentary, ALA supplemented‐sedentary, control diet‐exercise trained or ALA supplemented‐exercise trained. Following a 4 week intervention, although the independent and combined effects of ALA and exercise reduced (P

Key points
The role of trimeric intracellular cation (TRIC) channels is not known, although evidence suggests they may regulate ryanodine receptors (RyR) via multiple mechanisms. We therefore investigated whether Tric‐a gene knockout (KO) alters the single‐channel function of skeletal RyR (RyR1).
We find that RyR1 from Tric‐a KO mice are more sensitive to inhibition by divalent cations, although they respond normally to cytosolic Ca2+, ATP, caffeine and luminal Ca2+.
In the presence of Mg2+, ATP cannot effectively activate RyR1 from Tric‐a KO mice.
Additionally, RyR1 from Tric‐a KO mice are not activated by protein kinase A phosphorylation, demonstrating a defect in the ability of β‐adrenergic stimulation to regulate sarcoplasmic reticulum (SR) Ca2+‐release.
The defective RyR1 gating that we describe probably contributes significantly to the impaired SR Ca2+‐release observed in skeletal muscle from Tric‐a KO mice, further highlighting the importance of TRIC‐A for normal physiological regulation of SR Ca2+‐release in skeletal muscle.
Abstract
The type A trimeric intracellular cation channel (TRIC‐A) is a major component of the nuclear and sarcoplasmic reticulum (SR) membranes of cardiac and skeletal muscle, and is localized closely with ryanodine receptor (RyR) channels in the SR terminal cisternae. The skeletal muscle of Tric‐a knockout (KO) mice is characterized by Ca2+ overloaded and swollen SR and by changes in the properties of SR Ca2+ release. We therefore investigated whether RyR1 gating behaviour is modified in the SR from Tric‐a KO mice by incorporating native RyR1 into planar phospholipid bilayers under voltage‐clamp conditions. We find that RyR1 channels from Tric‐a KO mice respond normally to cytosolic Ca2+, ATP, adenine, caffeine and to luminal Ca2+. However, the channels are more sensitive to the inactivating effects of divalent cations, thus, in the presence of Mg2+, ATP is inadequate as an activator. Additionally, channels are not characteristically activated by protein kinase A even though the phosphorylation levels of Ser2844 are similar to controls. The results of the present study suggest that TRIC‐A functions as an excitatory modulator of RyR1 channels within the SR terminal cisternae. Importantly, this regulatory action of TRIC‐A appears to be independent of (although additive to) any indirect consequences to RyR1 activity that arise as a result of K+ fluxes across the SR via TRIC‐A.

Key points
Layer 2/3 neurons of the prefrontal cortex display higher gain of somatic excitability, responding with a higher number of action potentials for a given stimulus, in fmr1−/y mice.
In fmr1−/y L2/3 neurons, action potentials are taller, faster and narrower.
Outside‐out patch clamp recordings revealed that the maximum Na+ conductance density is higher in fmr1−/y L2/3 neurons.
Measurements of three biophysically distinct K+ currents revealed a depolarizing shift in the activation of a rapidly inactivating (A‐type) K+ conductance.
Realistic neuronal simulations of the biophysical observations recapitulated the elevated action potential and repetitive firing phenotype.
Abstract
Fragile X syndrome is the most common form of inherited mental impairment and autism. The prefrontal cortex is responsible for higher order cognitive processing, and prefrontal dysfunction is believed to underlie many of the cognitive and behavioural phenotypes associated with fragile X syndrome. We recently demonstrated that somatic and dendritic excitability of layer (L) 5 pyramidal neurons in the prefrontal cortex of the fmr1−/y mouse is significantly altered due to changes in several voltage‐gated ion channels. In addition to L5 pyramidal neurons, L2/3 pyramidal neurons play an important role in prefrontal circuitry, integrating inputs from both lower brain regions and the contralateral cortex. Using whole‐cell current clamp recording, we found that L2/3 pyramidal neurons in prefrontal cortex of fmr1−/y mouse fired more action potentials for a given stimulus compared with wild‐type neurons. In addition, action potentials in fmr1−/y neurons were significantly larger, faster and narrower. Voltage clamp of outside‐out patches from L2/3 neurons revealed that the transient Na+ current was significantly larger in fmr1−/y neurons. Furthermore, the activation curve of somatic A‐type K+ current was depolarized. Realistic conductance‐based simulations revealed that these biophysical changes in Na+ and K+ channel function could reliably reproduce the observed increase in action potential firing and altered action potential waveform. These results, in conjunction with our prior findings on L5 neurons, suggest that principal neurons in the circuitry of the medial prefrontal cortex are altered in distinct ways in the fmr1−/y mouse and may contribute to dysfunctional prefrontal cortex processing in fragile X syndrome.

Key points
In a rat model of ageing that is free of atherosclerosis or hypertension, E/A, a diagnostic measure of diastolic filling, decreases, and isovolumic relaxation time increases, indicating that both active and passive ventricular relaxation are impaired with advancing age.
Resting coronary blood flow and coronary functional hyperaemia are reduced with age, and endothelium‐dependent vasodilatation declines with age in coronary resistance arterioles.
Exercise training reverses age‐induced declines in diastolic and coronary microvascular function.
Thus, microvascular dysfunction and inadequate coronary perfusion are likely mechanisms of diastolic dysfunction in aged rats.
Exercise training, initiated at an advanced age, reverses age‐related diastolic and microvascular dysfunction; these data suggest that late‐life exercise training can be implemented to improve coronary perfusion and diastolic function in the elderly.
Abstract
The risk for diastolic dysfunction increases with advancing age. Regular exercise training ameliorates age‐related diastolic dysfunction; however, the underlying mechanisms have not been identified. We investigated whether (1) microvascular dysfunction contributes to the development of age‐related diastolic dysfunction, and (2) initiation of late‐life exercise training reverses age‐related diastolic and microvascular dysfunction. Young and old rats underwent 10 weeks of exercise training or remained as sedentary, cage‐controls. Isovolumic relaxation time (IVRT), early diastolic filling (E/A), myocardial performance index (MPI) and aortic stiffness (pulse wave velocity; PWV) were evaluated before and after exercise training or cage confinement. Coronary blood flow and vasodilatory responses of coronary arterioles were evaluated in all groups at the end of training. In aged sedentary rats, compared to young sedentary rats, a 42% increase in IVRT, a 64% decrease in E/A, and increased aortic stiffness (PWV: 6.36 ± 0.47 vs.4.89 ± 0.41, OSED vs. YSED, P

Key points
Leptin, is a 16 kDa pleiotropic peptide not only primarily secreted by adipocytes, but also produced by other tissues, including the heart.
Controversy exists regarding the adverse and beneficial effects of leptin on the heart
We analysed the effect of a non‐hypertensive dose of leptin on cardiac function, [Ca2+]i handling and cellular electrophysiology, which participate in the genesis of pump failure and related arrhythmias, both in control mice and in mice subjected to chronic pressure‐overload by transverse aorta constriction.
We find that leptin activates mechanisms that contribute to cardiac dysfunction under physiological conditions. However, after the establishment of pressure overload, an increase in leptin levels has protective cardiac effects with respect to rescuing the cellular heart failure phenotype.
These beneficial effects of leptin involve restoration of action potential duration via normalization of transient outward potassium current and sarcoplasmic reticulum Ca2+ content via rescue of control sarcoplasmic/endoplasmic reticulum Ca2+ ATPase levels and ryanodine receptor function modulation, leading to normalization of Ca2+ handling parameters.
Abstract
Leptin, is a 16 kDa pleiotropic peptide not only primary secreted by adipocytes, but also produced by other tissues, including the heart. Evidence indicates that leptin may have either adverse or beneficial effects on the heart. To obtain further insights, in the present study, we analysed the effect of leptin treatment on cardiac function, [Ca2+]i handling and cellular electrophysiology, which participate in the genesis of pump failure and related arrhythmias, both in control mice and in mice subjected to chronic pressure‐overload by transverse aorta constriction (TAC). Three weeks after surgery, animals received either leptin (0.36 mg kg–1 day–1) or vehicle via osmotic minipumps for 3 weeks. Echocardiographic measurements showed that, although leptin treatment was deleterious on cardiac function in sham, leptin had a cardioprotective effect following TAC. [Ca2+]i transient in cardiomyocytes followed similar pattern. Patch clamp experiments showed prolongation of action potential duration (APD) in TAC and leptin‐treated sham animals, whereas, following TAC, leptin reduced the APD towards control values. APD variations were associated with decreased transient outward potassium current and Kv4.2 and KChIP2 protein expression. TAC myocytes showed a higher incidence of triggered activities and spontaneous Ca2+ waves. These proarrhythmic manifestations, related to Ca2+/calmodulin‐dependent protein kinase II and ryanodine receptor phosphorylation, were reduced by leptin. The results of the present study demonstrate that, although leptin treatment was deleterious on cardiac function in control animals, leptin had a cardioprotective effect following TAC, normalizing cardiac function and reducing arrhythmogeneity at the cellular level.

Key points
A functional coupling between extrasynaptic NMDA receptors (eNMDARs) and the A‐type K+ current (IA) influences homeostatic firing responses of magnocellular neurosecretory cells (MNCs) to a physiological challenge. However, whether an altered eNMDAR–IA coupling also contributes to exacerbated MNC activity and neurohumoral activation during disease states is unknown.
We show that activation of eNMDARs by exogenously applied NMDA inhibited IA in MNCs obtained from sham, but not in MNCs from renovascular hypertensive (RVH) rats.
Neither the magnitude of the exogenously evoked NMDA current nor the expression of NMDAR subunits were altered in RVH rats.
Conversely, we found that a larger endogenous glutamate tone, which was not due to blunted glutamate transport activity, led to the sustained activation of eNMDARs that tonically inhibited IA, contributing in turn to higher firing activity in RVH rats.
Our studies show that exacerbated activation of eNMDARs by endogenous glutamate contributes to tonic inhibition of IA and enhanced MNC excitability in RVH rats.
Abstract
We recently showed that a functional coupling between extrasynaptic NMDA receptors (eNMDARs) and the A‐type K+ current (IA) influences the firing activity of hypothalamic magnocellular neurosecretory neurons (MNCs), as well as homeostatic adaptive responses to a physiological challenge. Here, we aimed to determine whether changes in the eNMDAR–IA coupling also contributed to exacerbated MNC activity during disease states. We used a combination of patch‐clamp electrophysiology and real‐time PCR in MNCs in sham and renovascular hypertensive (RVH) rats. Activation of eNMDARs by exogenously applied NMDA inhibited IA in sham rats, but this effect was largely blunted in RVH rats. The blunted response was not due to changes in eNMDAR expression and/or function, since neither NMDA current magnitude or reversal potential, nor the levels of NR1‐NR2A–D subunit expression were altered in RVH rats. Conversely, we found a larger endogenous glutamate tone, resulting in the sustained activation of eNMDARs that tonically inhibited IA and contributed also to higher ongoing firing activity in RVH rats. The enhanced endogenous glutamate tone in RVH rats was not due to blunted glutamate transporter activity. Rather, a higher transporter activity was observed, which possibly acted as a compensatory mechanism in the face of the elevated endogenous tone. In summary, our studies indicate that an elevated endogenous glutamate tone results in an exacerbated activation of eNMDARs, which in turn contributes to diminished IA magnitude and increased firing activity of MNCs from hypertensive rats.

Key points
Dentate spikes are fast fluctuations of hilar local‐field potentials that take place during rest and are thought to reflect input arriving from the entorhinal cortex to the hippocampus.
During dentate spikes, neuronal firing in hippocampal input (dentate gyrus) and output (CA1/CA3) regions is uncoupled.
To date, the behavioural significance of dentate spikes is unknown.
Here, we provide evidence that disrupting the dentate spike‐related uncoupling of the dentate gyrus and the CA1/CA3 subregions for 1 h after training retards associative learning.
We suggest dentate spikes play a significant role in memory consolidation.
Abstract
Hippocampal electrophysiological oscillations, namely theta and ripples, have been implicated in encoding and consolidation of new memories, respectively. According to existing literature, hippocampal dentate spikes are prominent, short‐duration (<30 ms), large‐amplitude (∼2–4 mV) fluctuations in hilar local‐field potentials that take place during awake immobility and sleep. Interestingly, previous studies indicate that during dentate spikes dentate gyrus granule cells increase their firing while firing of CA1 pyramidal cells are suppressed, thus resulting in momentary uncoupling of the two hippocampal subregions. To date, the behavioural significance of dentate spikes is unknown. Here, to study the possible role of dentate spikes in learning, we trained adult male Sprague–Dawley rats in trace eyeblink classical conditioning. For 1 h immediately following each conditioning session, one group of animals received hippocampal stimulation via the ventral hippocampal commissure (vHC) contingent on dentate spikes to disrupt the uncoupling between the dentate gyrus and the CA1 subregions. A yoked control group was stimulated during immobility, irrespective of brain state, and another control group was not stimulated at all. As a result, learning was impaired only in the group where vHC stimulation was administered contingent on dentate spikes. Our results suggest dentate spikes and/or the associated uncoupling of the dentate gyrus and the CA1 play a significant role in memory consolidation. Dentate spikes could possibly reflect reactivation and refinement of a memory trace within the dentate gyrus triggered by input from the entorhinal cortex.

Key points
Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia.
The extent of cross‐talk between fibroblasts, as the source of matrix protein, and satellite cells in humans is unknown. We studied this in human muscle biopsies and cell‐culture studies.
We observed a strong stimulation of myogenesis by human fibroblasts in cell culture.
In biopsies collected 30 days after a muscle injury protocol, fibroblast number increased to four times control levels, where fibroblasts were found to be preferentially located immediately surrounding regenerating muscle fibres.
These novel findings indicate an important role for fibroblasts in supporting the regeneration of muscle fibres, potentially through direct stimulation of satellite cell differentiation and fusion, and contribute to understanding of cell–cell cross‐talk during physiological and pathological muscle remodelling.
Abstract
Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. In addition to the indispensable role satellite cells play in muscle regeneration, there is emerging evidence in rodents for a regulatory influence on fibroblast activity. However, the influence of fibroblasts on satellite cells and muscle regeneration in humans is unknown. The purpose of this study was to investigate this in vitro and during in vivo regeneration in humans. Following a muscle injury protocol in young healthy men (n = 7), the number of fibroblasts (TCF7L2+), satellite cells (Pax7+), differentiating myogenic cells (myogenin+) and regenerating fibres (neonatal/embryonic myosin+) was determined from biopsy cross‐sections. Fibroblasts and myogenic precursor cells (MPCs) were also isolated from human skeletal muscle (n = 4) and co‐cultured using different cell ratios, with the two cell populations either in direct contact with each other or separated by a permeable membrane. MPC proliferation, differentiation and fusion were assessed from cells stained for BrdU, desmin and myogenin. On biopsy cross‐sections, fibroblast number was seen to increase, along with myogenic cell number, by d7 and increase further by d30, where fibroblasts were observed to be preferentially located immediately surrounding regenerating muscle fibres. In vitro, the presence of fibroblasts in direct contact with MPCs was found to moderately stimulate MPC proliferation and strongly stimulate both MPC differentiation and MPC fusion. It thus appears, in humans, that fibroblasts exert a strong positive regulatory influence on MPC activity, in line with observations during in vivo skeletal muscle regeneration.

Key points
A popular conception of mammalian cochlear physiology is that tuned mechanical vibration of the basilar membrane defines the frequency response of the innervating auditory nerve fibres
However, the data supporting these concepts come from vibratory measurements at cochlear locations tuned to high frequencies (>7 kHz).
Here, we measured the travelling wave in regions of the guinea pig cochlea that respond to low frequencies (<2 kHz) and found that mechanical tuning was broad and did not match auditory nerve tuning characteristics.
Non‐linear amplification of the travelling wave functioned over a broad frequency range and did not substantially sharpen frequency tuning.
Thus, the neural encoding of low‐frequency sounds, which includes most of the information conveyed by human speech, is not principally determined by basilar membrane mechanics.
Abstract
The popular notion of mammalian cochlear function is that auditory nerves are tuned to respond best to different sound frequencies because basilar membrane vibration is mechanically tuned to different frequencies along its length. However, this concept has only been demonstrated in regions of the cochlea tuned to frequencies >7 kHz, not in regions sensitive to lower frequencies where human speech is encoded. Here, we overcame historical technical limitations and non‐invasively measured sound‐induced vibrations at four locations distributed over the apical two turns of the guinea pig cochlea. In turn 3, the responses demonstrated low‐pass filter characteristics. In turn 2, the responses were low‐pass‐like, in that they occasionally did have a slight peak near the corner frequency. The corner frequencies of the responses were tonotopically tuned and ranged from 384 to 668 Hz. Non‐linear gain, or amplification of the vibrations in response to low‐intensity stimuli, was found both below and above the corner frequencies. Post mortem, cochlear gain disappeared. The non‐linear gain was typically 10–30 dB and was broad‐band rather than sharply tuned. However, the gain did reach nearly 50 dB in turn 2 for higher stimulus frequencies, nearly the amount of gain found in basal cochlear regions. Thus, our data prove that mechanical responses do not match neural responses and that cochlear amplification does not appreciably sharpen frequency tuning for cochlear regions that respond to frequencies <2 kHz. These data indicate that the non‐linear processing of sound performed by the guinea pig cochlea varies substantially between the cochlear apex and base.

Key points
At the cellular level cardiac hypertrophy causes remodelling, leading to changes in ionic channel, pump and exchanger densities and kinetics.
Previous studies have focused on quantifying changes in channels, pumps and exchangers without quantitatively linking these changes with emergent cellular scale functionality.
Two biophysical cardiac cell models were created, parameterized and validated and are able to simulate electrophysiology and calcium dynamics in myocytes from control sham operated rats and aortic‐banded rats exhibiting diastolic dysfunction.
The contribution of each ionic pathway to the calcium kinetics was calculated, identifying the L‐type Ca2+ channel and sarco/endoplasmic reticulum Ca2+ATPase as the principal regulators of systolic and diastolic Ca2+, respectively.
Results show that the ability to dynamically change systolic Ca2+, through changes in expression of key Ca2+ modelling protein densities, is drastically reduced following the aortic banding procedure; however the cells are able to compensate Ca2+ homeostasis in an efficient way to minimize systolic dysfunction.
Abstract
Elevated left ventricular afterload leads to myocardial hypertrophy, diastolic dysfunction, cellular remodelling and compromised calcium dynamics. At the cellular scale this remodelling of the ionic channels, pumps and exchangers gives rise to changes in the Ca2+ transient. However, the relative roles of the underlying subcellular processes and the positive or negative impact of each remodelling mechanism are not fully understood. Biophysical cardiac cell models were created to simulate electrophysiology and calcium dynamics in myocytes from control rats (SHAM) and aortic‐banded rats exhibiting diastolic dysfunction. The model parameters and framework were validated and the fitted parameters demonstrated to be unique for explaining our experimental data. The contribution of each ionic pathway to the calcium kinetics was calculated, identifying the L‐type Ca2+ channel (LCC) and the sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) as the principal regulators of systolic and diastolic Ca2+, respectively. In the aortic banding model, the sensitivity of systolic Ca2+ to LCC density and diastolic Ca2+ to SERCA density decreased by 16‐fold and increased by 23%, respectively, relative to the SHAM model. The energy cost of ionic homeostasis is maintained across the two models. The models predict that changes in ionic pathway densities in compensated aortic banding rats maintain Ca2+ function and efficiency. The ability to dynamically alter systolic function is significantly diminished, while the capacity to maintain diastolic Ca2+ is moderately increased.

Key points
Maternal nutrient restriction induces intrauterine growth restriction (IUGR) and leads to heightened cardiovascular risks later in life.
We report right ventricular (RV) filling and ejection abnormalities in IUGR young adult baboons using cardiac magnetic resonance imaging.
Both functional and morphological indicators of poor RV function were seen, many of which were similar to effects of ageing, but also with a few key differences.
We observed more pronounced RV changes compared to our previous report of the left ventricle, suggesting there is likely to be a component of isolated RV abnormality in addition to expected haemodynamic sequelae from left ventricular dysfunction. In particular, our findings raise the suspicion of pulmonary hypertension after IUGR.
This study establishes that IUGR also leads to impairment of the right ventricle in addition to the left ventricle classically studied.
Abstract
Maternal nutrient restriction induces intrauterine growth restriction (IUGR), increasing later life chronic disease including cardiovascular dysfunction. Our left ventricular (LV) CMRI studies in IUGR baboons (8 M, 8 F, 5.7 years – human equivalent approximately 25 years), control offspring (8 M, 8 F, 5.6 years), and normal elderly (OLD) baboons (6 M, 6 F, mean 15.9 years) revealed long‐term LV abnormalities in IUGR offspring. Although it is known that right ventricular (RV) function is dependent on LV health, the IUGR right ventricle remains poorly studied. We examined the right ventricle with cardiac magnetic resonance imaging in the same cohorts. We observed decreased ejection fraction (49 ± 2 vs. 33 ± 3%, P

An important principle in the organization of the somatosensory cortex is that it processes afferent information from the contralateral side of the body. The role of the ipsilateral somatosensory cortex (iS1) in sensory gating in humans remains largely unknown. Using electroencephalographic recordings over the iS1 and electrical stimulation of the ulnar nerve at the wrist we examined somatosensory evoked potentials (SSEPs; P14/N20, N20/P25, and P25/N33 components) and paired‐pulse SSEPs between (interhemispheric inhibition) and within (intracortical inhibition) the iS1 at rest and during tonic index finger voluntary activity. We found that the amplitude of the P25/N33, but not other SSEP components, was reduced during voluntary activity compared with rest. Interhemispheric inhibition increased the amplitude of the P25/N33 and intracortical inhibition reduced the amplitude of the P25/N33, suggesting a cortical origin for this effect. The P25/N33 receives inputs from the motor cortex, therefore, we also examined the contribution of distinct sets of cortical interneurons by testing the effect of ulnar nerve stimulation on motor evoked potentials (MEPs) elicited by transcranial magnetic stimulation over the ipsilateral motor cortex with the coil in the posterior‐anterior (PA) and anterior‐posterior (AP) orientation. Afferent input attenuated PA, but not AP, MEPs during voluntary activity compared with rest. Notably, changes in interhemispheric inhibition correlated with changes in PA MEPs. Our novel findings suggest that interhemispheric projections between S1s and intracortical circuits, likely from somatosensory and motor cortex, contribute to sensory gating in the iS1 during voluntary activity in humans.
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Key points
Afterhyperpolarizations (AHPs) generated by repetitive action potentials in supraoptic magnocellular neurons regulate repetitive firing and spike frequency adaptation but relatively little is known about PIP2’s control of these AHPs.
We examined how changes in PIP2 levels affected AHPs, somatic [Ca2+]i, and whole cell Ca2+ currents.
Manipulations of PIP2 levels affected both medium and slow AHP currents in oxytocin (OT) neurons of the supraoptic nucleus.
Manipulations of PIP2 levels did not modulate AHPs by influencing Ca2+ release from IP3‐triggered Ca2+ stores, suggesting more direct modulation of channels by PIP2.
PIP2 depletion reduced spike‐evoked Ca2+ entry and voltage‐gated Ca2+ currents.
PIP2 appears to influence AHPs in OT neurons by reducing Ca2+ influx during spiking.
Abstract
Oxytocin (OT)‐ and vasopressin (VP)‐secreting magnocellular neurons of the supraoptic nucleus (SON) display calcium‐dependent afterhyperpolarizations (AHPs) following a train of action potentials that are critical to shaping the firing patterns of these cells. Previous work demonstrated that the lipid phosphatidylinositol 4,5‐bisphosphate (PIP2) enabled the slow AHP component (sAHP) in cortical pyramidal neurons. We investigated whether this phenomenon occurred in OT and VP neurons of the SON. Using whole cell recordings in coronal hypothalamic slices from adult female rats, we demonstrated that inhibition of PIP2 synthesis with wortmannin robustly blocked both the medium and slow AHP currents (ImAHP and IsAHP) of OT, but not VP neurons with high affinity. We further tested this by introducing a water‐soluble PIP2 analogue (diC8‐PIP2) into neurons, which in OT neurons not only prevented wortmannin's inhibitory effect, but slowed rundown of the ImAHP and IsAHP. Inhibition of phospholipase C (PLC) with U73122 did not inhibit either ImAHP or IsAHP in OT neurons, consistent with wortmannin's effects not being due to reducing diacylglycerol (DAG) or IP3 availability, i.e. PIP2 modulation of AHPs is not likely to involve downstream Ca2+ release from inositol 1,4,5‐trisphosphate (IP3)‐triggered Ca2+‐store release, or channel modulation via DAG and protein kinase C (PKC). We found that wortmannin reduced [Ca2+]i increase induced by spike trains in OT neurons, but had no effect on AHPs evoked by uncaging intracellular Ca2+. Finally, wortmannin selectively reduced whole cell Ca2+ currents in OT neurons while leaving VP neurons unaffected. The results indicate that PIP2 modulates both the ImAHP and IsAHP in OT neurons, most likely by controlling Ca2+ entry through voltage‐gated Ca2+ channels opened during spike trains.

Key points
The prevalence of cardiorespiratory fitness (CRF) non‐response gradually declines in healthy individuals exercising 60, 120, 180, 240 or 300 min per week for 6 weeks.
Following a successive identical 6‐week training period but comprising 120 min of additional exercise per week, CRF non‐response is universally abolished.
The magnitude of CRF improvement is primarily attributed to changes in haemoglobin mass.
The potential for CRF improvement may be present and unveiled with appropriate exercise training stimuli in healthy individuals without exception.
Abstract
One in five adults following physical activity guidelines are reported to not demonstrate any improvement in cardiorespiratory fitness (CRF). Herein, we sought to establish whether CRF non‐response to exercise training is dose‐dependent, using a between‐ and within‐subject study design. Seventy‐eight healthy adults were divided into five groups (1–5) respectively comprising one, two, three, four and five 60 min exercise sessions per week but otherwise following an identical 6‐week endurance training (ET) programme. Non‐response was defined as any change in CRF, determined by maximal incremental exercise power output (Wmax), within the typical error of measurement (±3.96%). Participants classified as non‐responders after the ET intervention completed a successive 6‐week ET period including two additional exercise sessions per week. Maximal oxygen consumption (V̇O2 max ), haematology and muscle biopsies were assessed prior to and after each ET period. After the first ET period, Wmax increased (P

Key points
Mammalian cells are frequently exposed to stressors causing volume changes.
The transient receptor potential vanilloid 4 (TRPV4) channel translates osmotic stress into ion flux.
The molecular mechanism coupling osmolarity to TRPV4 activation remains elusive.
TRPV4 responds to isosmolar cell swelling and osmolarity translated via different aquaporins.
TRPV4 functions as a volume‐sensing ion channel irrespective of the origin of the cell swelling.
Abstract
Transient receptor potential channel 4 of the vanilloid subfamily (TRPV4) is activated by a diverse range of molecular cues, such as heat, lipid metabolites and synthetic agonists, in addition to hyposmotic challenges. As a non‐selective cation channel permeable to Ca2+, it transduces physical stress in the form of osmotic cell swelling into intracellular Ca2+‐dependent signalling events. Its contribution to cell volume regulation might include interactions with aquaporin (AQP) water channel isoforms, although the proposed requirement for a TRPV4–AQP4 macromolecular complex remains to be resolved. To characterize the elusive mechanics of TRPV4 volume‐sensing, we expressed the channel in Xenopus laevis oocytes together with AQP4. Co‐expression with AQP4 facilitated the cell swelling induced by osmotic challenges and thereby activated TRPV4‐mediated transmembrane currents. Similar TRPV4 activation was induced by co‐expression of a cognate channel, AQP1. The level of osmotically‐induced TRPV4 activation, although proportional to the degree of cell swelling, was dependent on the rate of volume changes. Importantly, isosmotic cell swelling obtained by parallel activation of the co‐expressed water‐translocating Na+/K+/2Cl− cotransporter promoted TRPV4 activation despite the absence of the substantial osmotic gradients frequently employed for activation. Upon simultaneous application of an osmotic gradient and the selective TRPV4 agonist GSK1016790A, enhanced TRPV4 activation was observed only with subsaturating stimuli, indicating that the agonist promotes channel opening similar to that of volume‐dependent activation. We propose that, contrary to the established paradigm, TRPV4 is activated by increased cell volume irrespective of the molecular mechanism underlying cell swelling. Thus, the channel functions as a volume‐sensor, rather than as an osmo‐sensor.

Key points
Changes in nerve conduction velocity following an impulse (i.e. velocity recovery cycles) reflect after‐potentials, and can provide an indication of altered nerve membrane properties.
This study used microneurography to assess the effects of ischaemia on single human sympathetic fibres innervating the dorsum of the foot.
It was found that velocity recovery cycles can distinguish whether a sympathetic nerve fibre is depolarized or not.
The method may be used to detect membrane depolarization of sympathetic nerve fibres in human patients when autonomic neuropathy is suspected.
Abstract
The aim of this study was to determine whether velocity recovery cycles (VRCs) could detect the effects of ischaemia on sympathetic nerve fibres. VRCs of human sympathetic nerve fibres of the superficial peroneal nerve innervating the dorsum of the foot were recorded by microneurography in seven healthy volunteers. Sympathetic nerve fibres were identified by studying their response to manoeuvres increasing sympathetic outflow and by measuring activity‐dependent slowing at 2 Hz stimulation. VRCs were assessed at rest, during 30 min of induced limb ischaemia and during 20 min of recovery after ischaemia. From each VRC was measured the relative refractory period (RRP), the supernormality and the time to peak supernormality (SN@). During ischaemia, RRP increased from the baseline value of 37.4 ± 8.7 ms (mean ± SEM) to 67.1 ± 12.1 ms (P

Key points
Heart size increases with age (called hypertrophy), and its ability to contract declines. However, these reflect average changes that may not be present, or present to the same extent, in all older individuals.
That aging happens at different rates is well accepted clinically. People who are aging rapidly are frail and frailty is measured with a ‘frailty index’.
We quantified frailty with a validated mouse frailty index tool and evaluated the impacts of age and frailty on cardiac hypertrophy and contractile dysfunction.
Hypertrophy increased with age, while contractions, calcium currents and calcium transients declined; these changes were graded by frailty scores.
Overall health status, quantified as frailty, may promote maladaptive changes associated with cardiac aging and facilitate the development of diseases such as heart failure.
To understand age‐related changes in heart structure and function, it is essential to know both chronological age and the health status of the animal.
Abstract
On average, cardiac hypertrophy and contractile dysfunction increase with age. Still, individuals age at different rates and their health status varies from fit to frail. We investigated the influence of frailty on age‐dependent ventricular remodelling. Frailty was quantified as deficit accumulation in adult (≈7 months) and aged (≈27 months) C57BL/6J mice by adapting a validated frailty index (FI) tool. Hypertrophy and contractile function were evaluated in Langendorff‐perfused hearts; cellular correlates/mechanisms were investigated in ventricular myocytes. FI scores increased with age. Mean cardiac hypertrophy increased with age, but values in the adult and aged groups overlapped. When plotted as a function of frailty, hypertrophy was graded by FI score (r = 0.67–0.55, P

Key points
In the heart, endothelial nitric oxide (NO) controls oxygen consumption in the working heart through paracrine mechanisms.
While cardiac myocytes contain several isoforms of NO synthases, it is unclear whether these can control respiration in an intracrine fashion.
A long‐standing controversy is whether a NOS exists within mitochondria.
By combining fluorescence technologies with electrical field stimulation or the patch‐clamp technique in beating cardiac myocytes, we identified a neuronal NO synthase (nNOS) as the most relevant source of intracellular NO during β‐adrenergic stimulation, while no evidence for a mitochondria‐located NOS was obtained.
The amounts of NO produced by non‐mitochondrial nNOS were insufficient to regulate respiration during β‐adrenergic stimulation, arguing against intracrine control of respiration by NO within cardiac myocytes.
Abstract
Endothelial nitric oxide (NO) controls cardiac oxygen (O2) consumption in a paracrine way by slowing respiration at the mitochondrial electron transport chain. While NO synthases (NOSs) are also expressed in cardiac myocytes, it is unclear whether they control respiration in an intracrine way. Furthermore, the existence of a mitochondrial NOS is controversial. Here, by combining fluorescence imaging with electrical field stimulation, the patch‐clamp method and knock‐out technology, we determined the sources and consequences of intracellular NO formation during workload transitions in isolated murine and guinea pig cardiac myocytes and mitochondria. Using 4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate (DAF) as a fluorescent NO‐sensor that locates to the cytosol and mitochondria, we observed that NO increased by ∼12% within 3 min of β‐adrenergic stimulation in beating cardiac myocytes. This NO stems from neuronal NOS (nNOS), but not endothelial (eNOS). After patch clamp‐mediated dialysis of cytosolic DAF, the remaining NO signals (mostly mitochondrial) were blocked by nNOS deletion, but not by inhibiting the mitochondrial Ca2+ uniporter with Ru360. While in isolated mitochondria exogenous NO inhibited respiration and reduced the NAD(P)H redox state, pyridine nucleotide redox states were unaffected by pharmacological or genetic disruption of endogenous nNOS or eNOS during workload transitions in cardiac myoctyes. We conclude that under physiological conditions, nNOS is the most relevant source for NO in cardiac myocytes, but this nNOS is not located in mitochondria and does not control respiration. Therefore, cardiac O2 consumption is controlled by endothelial NO in a paracrine, but not intracrine, fashion.

Key points
A subpopulation of retinal ganglion cells expresses the neuropeptide vasopressin.
These retinal ganglion cells project predominately to our biological clock, the suprachiasmatic nucleus (SCN).
Light‐induced vasopressin release enhances the responses of SCN neurons to light.
It also enhances expression of genes involved in photo‐entrainment of biological rhythms.
Abstract
In all animals, the transition between night and day engages a host of physiological and behavioural rhythms. These rhythms depend not on the rods and cones of the retina, but on retinal ganglion cells (RGCs) that detect the ambient light level in the environment. These project to the suprachiasmatic nucleus (SCN) of the hypothalamus to entrain circadian rhythms that are generated within the SCN. The neuropeptide vasopressin has an important role in this entrainment. Many SCN neurons express vasopressin, and it has been assumed that the role of vasopressin in the SCN reflects the activity of these cells. Here we show that vasopressin is also expressed in many retinal cells that project to the SCN. Light‐evoked vasopressin release contributes to the responses of SCN neurons to light, and enhances expression of the immediate early gene c‐fos in the SCN, which is involved in photic entrainment of circadian rhythms.

Key points
Repolarizing currents through K+ channels are essential for proper sinoatrial node (SAN) pacemaking, but the influence of intracellular Ca2+ on repolarization in the SAN is uncertain.
We identified all three isoforms of Ca2+‐activated small conductance K+ (SK) channels in the murine SAN.
SK channel blockade slows repolarization and subsequent depolarization of SAN cells.
In the atrial‐specific Na+/Ca2+ exchanger (NCX) knockout mouse, cellular Ca2+ accumulation during spontaneous SAN pacemaker activity produces intermittent hyperactivation of SK channels, leading to arrhythmic pauses alternating with bursts of pacing.
These findings suggest that Ca2+‐sensitive SK channels can translate changes in cellular Ca2+ into a repolarizing current capable of modulating pacemaking.
SK channels are a potential pharmacological target for modulating SAN rate or treating SAN dysfunction, particularly under conditions characterized by abnormal increases in diastolic Ca2+.
Abstract
Small conductance K+ (SK) channels have been implicated as modulators of spontaneous depolarization and electrical conduction that may be involved in cardiac arrhythmia. However, neither their presence nor their contribution to sinoatrial node (SAN) pacemaker activity has been investigated. Using quantitative PCR (q‐PCR), immunostaining and patch clamp recordings of membrane current and voltage, we identified all three SK isoforms (SK1, SK2 and SK3) in mouse SAN. Inhibition of SK channels with the specific blocker apamin prolonged action potentials (APs) in isolated SAN cells. Apamin also slowed diastolic depolarization and reduced pacemaker rate in isolated SAN cells and intact tissue. We investigated whether the Ca2+‐sensitive nature of SK channels could explain arrhythmic SAN pacemaker activity in the atrial‐specific Na+/Ca2+ exchange (NCX) knockout (KO) mouse, a model of cellular Ca2+ overload. SAN cells isolated from the NCX KO exhibited higher SK current than wildtype (WT) and apamin prolonged their APs. SK blockade partially suppressed the arrhythmic burst pacing pattern of intact NCX KO SAN tissue. We conclude that SK channels have demonstrable effects on SAN pacemaking in the mouse. Their Ca2+‐dependent activation translates changes in cellular Ca2+ into a repolarizing current capable of modulating regular pacemaking. This Ca2+ dependence also promotes abnormal automaticity when these channels are hyperactivated by elevated Ca2+. We propose SK channels as a potential target for modulating SAN rate, and for treating patients affected by SAN dysfunction, particularly in the setting of Ca2+ overload.

Key points
Large conductance, Ca2+‐activated K+ (BKCa) channels play important roles in mammalian retinal neurons, including photoreceptors, bipolar cells, amacrine cells and ganglion cells, but they have not been identified in horizontal cells.
BKCa channel blockers paxilline and iberiotoxin, as well as Ca2+ free solutions and divalent cation Cav channel blockers, eliminate the outwardly rectifying current, while NS1619 enhances it. In symmetrical 150 mm K+, single channels had a conductance close to 250 pS, within the range of all known BKCa channels.
In current clamped horizontal cells, BKCa channels subdue depolarizing membrane potential excursions, reduce the average resting potential and decrease oscillations. The results show that BKCa channel activation puts a ceiling on horizontal cell depolarization and regulates the temporal responsivity of the cells.
Abstract
Large conductance, calcium‐activated potassium (BKCa) channels have numerous roles in neurons including the regulation of membrane excitability, intracellular [Ca2+] regulation, and neurotransmitter release. In the retina, they have been identified in photoreceptors, bipolar cells, amacrine cells and ganglion cells, but have not been conclusively identified in mammalian horizontal cells. We found that outward current recorded between −30 and +60 mV is carried primarily in BKCa channels in isolated horizontal cells of rats and mice. Whole‐cell outward currents were maximal at +50 mV and declined at membrane potentials positive to this value. This current was eliminated by the selective BKCa channel blocker paxilline (100 nm), iberiotoxin (10 μm), Ca2+ free solutions and divalent cation Cav channel blockers. It was activated by the BKCa channel activator NS1619 (30 μm). Single channel recordings revealed the conductance of the channels to be 244 ± 11 pS (n = 17; symmetrical 150 mm K+) with open probability being both voltage‐ and Ca2+‐dependent. The channels showed fast activation kinetics in response to Ca2+ influx and inactivation gating that could be modified by intracellular protease treatment, which suggests β subunit involvement. Under current clamp, block of BKCa current increased depolarizing membrane potential excursions, raising the average resting potential and producing oscillations. BKCa current activation with NS1619 inhibited oscillations and hyperpolarized the resting potential. These effects underscore the functional role of BKCa current in limiting depolarization of the horizontal cell membrane potential and suggest actions of these channels in regulating the temporal responsivity of the cells.

Key points
Actomyosin ATP hydrolysis occurring during muscle contraction releases inorganic phosphate [Pi] in the myoplasm. High [Pi] reduces force and affects force kinetics in skinned muscle fibres at low temperature. These effects decrease at high temperature, raising the question of their importance under physiological conditions.
This study provides the first analysis of the effects of Pi on muscle performance in intact mammalian fibres at physiological temperature. Myoplasmic [Pi] was raised by fatiguing the fibres with a series of tetanic contractions.
[Pi] increase reduces muscular force mainly by decreasing the force of the single molecular motor, the crossbridge, and alters the crossbridge response to fast length perturbation indicating faster kinetics.
These results are in agreement with schemes of actomyosin ATPase and the crossbridge cycle including a low‐ or no‐force state and show that fibre length changes perturb the Pi‐sensitive force generation of the crossbridge cycle.
Abstract
Actomyosin ATP hydrolysis during muscle contraction releases inorganic phosphate, increasing [Pi] in the myoplasm. Experiments in skinned fibres at low temperature (10–12°C) have shown that [Pi] increase depresses isometric force and alters the kinetics of actomyosin interaction. However, the effects of Pi decrease with temperature and this raises the question of the role of Pi under physiological conditions. The present experiments were performed to investigate this point. Intact fibre bundles isolated from the flexor digitorum brevis of C57BL/6 mice were stimulated with a series of tetanic contractions at 1.5 s intervals at 33°C. As show previously the most significant change induced by a bout of contractile activity similar to the initial 10 tetani of the series was an increase of [Pi] without significant Ca2+ or pH changes. Measurements of force, stiffness and responses to fast stretches and releases were therefore made on the 10th tetanus of the series and compared with control. We found that (i) tetanic force at the 10th tetanus was ∼20% smaller than control without a significant decrease of crossbridge stiffness; and (ii) the force recovery following quick stretches and releases was faster than in control. These results indicate that at physiological temperature the increase of [Pi] occurring during early fatigue reduces tetanic force mainly by depressing the individual crossbridge force and accelerating crossbridge kinetics.

Key points
Chronic fetal hypoxaemia is a common pregnancy complication associated with intrauterine growth restriction that may influence respiratory outcome at birth.
We investigated the effect of maternal chronic hypoxia for a month in late gestation on signalling pathways regulating fetal lung maturation and the transition to air‐breathing at birth using isobaric hypoxic chambers without alterations to maternal food intake.
Maternal chronic hypoxia in late gestation increases fetal lung expression of genes regulating hypoxia signalling, lung liquid reabsorption and surfactant maturation, which may be an adaptive response in preparation for the successful transition to air‐breathing at birth.
In contrast to other models of chronic fetal hypoxaemia, late gestation onset fetal hypoxaemia promotes molecular regulation of fetal lung maturation. This suggests a differential effect of timing and duration of fetal chronic hypoxaemia on fetal lung maturation, which supports the heterogeneity observed in respiratory outcomes in newborns following exposure to chronic hypoxaemia in utero.
Abstract
Chronic fetal hypoxaemia is a common pregnancy complication that may arise from maternal, placental and/or fetal factors. Respiratory outcome of the infant at birth likely depends on the duration, timing and severity of the hypoxaemic insult. We have isolated the effect of maternal chronic hypoxia (MCH) for a month in late gestation on fetal lung development. Pregnant ewes were exposed to normoxia (21% O2) or hypoxia (10% O2) from 105 to 138 days of gestation (term ∼145 days). At 138 days, gene expression in fetal lung tissue was determined by quantitative RT‐PCR. Cortisol concentrations were determined in fetal plasma and lung tissue. Numerical density of surfactant protein positive cells was determined by immunohistochemistry. MCH reduced maternal PaO2 (106 ± 2.9 vs. 47 ± 2.8 mmHg) and fetal body weight (4.0 ± 0.4 vs. 3.2 ± 0.9 kg). MCH increased fetal lung expression of the anti‐oxidant marker CAT and decreased expression of the pro‐oxidant marker NOX‐4. MCH increased expression of genes regulating hypoxia signalling and feedback (HIF‐3α, KDM3A, SLC2A1, EGLN‐3). There was no effect of MCH on fetal plasma/lung tissue cortisol concentrations, nor genes regulating glucocorticoid signalling (HSD11B‐1, HSD11B‐2, NR3C1, NR3C2). MCH increased expression of genes regulating sodium (SCNN1‐B, ATP1‐A1, ATP1‐B1) and water (AQP‐4) movement in the fetal lung. MCH promoted surfactant maturation (SFTP‐B, SFTP‐D, ABCA3) at the molecular level, but did not alter the numerical density of surfactant positive cells in lung tissue. MCH in late gestation promotes molecular maturation of the fetal lung, which may be an adaptive response in preparation for the successful transition to air‐breathing at birth.

Key points
Golgi tendon organs (GTOs) and associated Ib reflexes contribute to standing balance, but the potential impacts of threats to standing balance on Ib reflexes are unknown.
Tendon electrical stimulation to the Achilles’ tendon was used to probe changes in Ib inhibition in medial gastrocnemius with postural orientation (lying prone vs. upright standing; experiment 1) and height‐induced postural threat (standing at low and high surface heights; experiment 2).
Ib inhibition was reduced while participants stood upright, compared to lying prone (42.2%); and further reduced when standing in the high, compared to low, threat condition (32.4%).
These experiments will impact future research because they demonstrate that tendon electrical stimulation can be used to probe Ib reflexes in muscles engaged in standing balance.
These results provide novel evidence that human short‐latency GTO‐Ib reflexes are dependent upon both task, as evidenced by changes with postural orientation, and context, such as height‐induced postural threat during standing.
Abstract
Golgi tendon organ Ib reflexes are thought to contribute to standing balance control, but it is unknown if they are modulated when people are exposed to a postural threat. We used a novel application of tendon electrical stimulation (TStim) to elicit Ib inhibitory reflexes in the medial gastrocnemius, while actively engaged in upright standing balance, to examine (a) how Ib reflexes to TStim are influenced by upright stance, and (b) the effects of height‐induced postural threat on Ib reflexes during standing. TStim evoked short‐latency (<47 ms) inhibition apparent in trigger‐averaged rectified EMG, which was quantified in terms of area, duration and mean amplitude of inhibition. In order to validate the use of TStim in a standing model, TStim‐Ib inhibition was compared from conditions where participants were lying prone vs. standing upright. TStim evoked Ib inhibition in both conditions; however, significant reductions in Ib inhibition area (42.2%) and duration (32.9%) were observed during stance. Postural threat, manipulated by having participants stand at LOW (0.8 m high, 0.6 m from edge) and HIGH (3.2 m, at edge) elevated surfaces, significantly reduced Ib inhibition area (32.4%), duration (16.4%) and amplitude (24.8%) in the HIGH, compared to LOW, threat condition. These results demonstrate TStim is a viable technique for investigating Ib reflexes in standing, and confirm Ib reflexes are modulated with postural orientation. The novel observation of reduced Ib inhibition with elevated postural threat reveals that human Ib reflexes are context dependent, and the human Ib reflex pathways are modulated by threat or emotional processing centres of the CNS.

Key points
Sensory neurons that innervate the bone marrow provide the CNS with information about pain associated with bone disease and pathology, but little is known of their function.
Here we use a novel in vivo bone–nerve electrophysiological preparation to study how they respond to noxious mechanical stimulation delivered by increasing intra‐osseous pressure.
We provide evidence that sensory neurons that innervate the bone marrow respond to high threshold noxious mechanical stimulation, have response properties consistent with a role in nociception, provide information about different features of an intra‐osseous pressure stimulus and express the Piezo2 mechano‐transducer molecule.
Our findings show how some bone marrow nociceptors signal pain in bony diseases and pathologies that involve a mechanical disturbance or increased intra‐osseous pressure, and that the Piezo2 mechano‐transducer may be involved.
Abstract
Whilst the sensory neurons and nerve terminals that innervate bone marrow have a morphology and molecular phenotype consistent with a role in nociception, little is known about their physiology or the mechanisms that generate and maintain bone pain. In the present study, we provide evidence that Aδ nociceptors that innervate the bone marrow respond to high threshold noxious mechanical stimulation, exhibit fatigue in response to prior stimulation and in some cases can be sensitized by capsaicin. They can be classified on the basis of their response properties as either phasic–tonic units that appear to code for different intensities of intra‐osseous pressure, or phasic units that code for the rate of change in intra‐osseous pressure. Three different subclasses of mechanically sensitive Aδ units were observed: phasic units that were sensitized by capsaicin, phasic units that were not sensitized by capsaicin and phasic–tonic units (that were not sensitized by capsaicin). These could also, in part, be distinguished by differences in their thresholds for activation, mean discharge frequency, latency to peak activation and peak‐to‐peak action potential amplitude. The majority of small‐diameter myelinated sensory neurons projecting to the bone marrow expressed Piezo2. Our findings indicate that Aδ mechano‐nociceptors are likely to play an important role in generating and maintaining pain in response to bony pathologies that involve a mechanical disturbance or increased intra‐osseous pressure, and imply that Piezo2 signalling may be involved in mechano‐transduction in these receptors.

Perinatal exposure to excess maternal intake of added sugars, including fructose and sucrose, is associated with an increased risk of obesity and type 2 diabetes in adult life. However, it is unknown to what extent the type of sugar and the timing of exposure affect these outcomes. The aim of this study was to determine the impact of exposure to maternal consumption of a 10% w/v beverage containing sucrose or high fructose corn syrup‐55 (HFCS‐55) during the prenatal and/or suckling periods on offspring at 3 and 12 weeks, utilising a cross‐fostering approach in a rodent model. Perinatal sucrose exposure decreased plasma glucose concentrations in offspring at 3 weeks, but did not alter glucose tolerance. Increased adiposity was observed in 3‐week‐old offspring exposed to sucrose or HFCS‐55 during suckling, with increased hepatic fat content in HFCS‐55‐exposed offspring. In terms of specific fatty acids, hepatic monounsaturated (omega‐7 and ‐9) fatty acid content was elevated at weaning, and was most pronounced in sucrose offspring exposed during both the prenatal and suckling periods, and HFCS‐55 offspring exposed during suckling only. By 12 weeks, the effects on adiposity and hepatic lipid composition were largely normalised. However, exposure to either sucrose or HFCS‐55 during the prenatal period only was associated with elevated plasma free fatty acids at weaning, and this effect persisted until 12 weeks. This study suggests that the type of sugar and the timing of exposure (prenatal or suckling periods) are both important for determining the impact on metabolic health outcomes in the offspring.
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Key points
Mutations in the gene encoding poly(A)‐binding protein nuclear 1 (PABPN1) result in oculopharyngeal muscular dystrophy (OPMD). This disease is of late‐onset, but the underlying mechanism is unclear.
Ca2+ stimulates muscle growth and contraction and, because OPMD courses with muscle atrophy and weakness, we hypothesized that the homeostasis of Ca2+ is altered in this disorder.
C2C12 myotubes were transfected with cDNAs encoding either PABPN1 or the PABPN1‐17A OPMD mutation. Subsequently, they were investigated concerning not only excitation–contraction coupling (ECC) and intracellular levels of Ca2+, but also differentiation stage and nuclear structure.
PABPN1‐17A gave rise to: inhibition of Ca2+ release during ECC, depletion of sarcoplasmic reticulum Ca2+ content, reduced expression of ryanodine receptors, altered nuclear morphology and incapability to stimulate myoblast fusion.
PABPN1‐17A failed to inhibit ECC in adult muscle fibres, suggesting that its effects are primarily related to muscle regeneration.
Abstract
Oculopharyngeal muscular dystrophy (OPMD) is linked to mutations in the gene encoding poly(A)‐binding protein nuclear 1 (PABPN1). OPMD mutations consist of an expansion of a tract that contains 10 alanines (to 12–17). This disease courses with muscle weakness that begins in adulthood, but the underlying mechanism is unclear. In the present study, we investigated the functional effects of PABPN1 and an OPMD mutation (PABPN1‐17A) using myotubes transfected with cDNAs encoding these proteins (GFP‐tagged). PABPN1 stimulated myoblast fusion (100%), whereas PABPN1‐17A failed to mimic this effect. Additionally, the OPMD mutation markedly altered nuclear morphology; specifically, it led to nuclei with a more convoluted and ovoid shape. Although PABPN1 and PABPN1‐17A modified the expression of sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase and calsequestrin, the corresponding changes did not have a clear impact on [Ca2+]. Interestingly, neither L‐type Ca2+ channels, nor voltage‐gated sarcoplasmic reticulum (SR) Ca2+ release (VGCR) was altered by PABPN1. However, PABPN1‐17A produced a selective inhibition of VGCR (50%). This effect probably arises from both lower expression of RyR1 and depletion of SR Ca2+. The latter, however, was not related to inhibition of store‐operated Ca2+ entry. Both PABPN1 constructs promoted a moderated decrease in cytosolic [Ca2+], which apparently results from down‐regulation of excitation‐coupled Ca2+ entry. On the other hand, PABPN1‐17A did not alter ECC in muscle fibres, suggesting that adult muscle is less prone to developing deleterious effects. These results demonstrate that PABPN1 proteins regulate essential processes during myotube formation and support the notion that OPMD involves disruption of myogenesis, nuclear structure and homeostasis of Ca2+.

Key points
The nucleus reuniens (Re), a nucleus of the midline thalamus, is part of a cognitive network including the hippocampus and the medial prefrontal cortex. To date, very few studies have examined the electrophysiological properties of Re neurons at a cellular level.
The majority of Re neurons exhibit spontaneous action potential firing at rest. This is independent of classical amino‐acid mediated synaptic transmission. When driven by various forms of depolarizing current stimulus, Re neurons display considerable diversity in their firing patterns. As a result of the presence of a low threshold Ca2+ channel, spike output functions are strongly modulated by the prestimulus membrane potential.
Finally, we describe a novel form of activity‐dependant intrinsic plasticity that eliminates the high‐frequency burst firing present in many Re neurons.
These results provide a comprehensive summary of the intrinsic electrophysiological properties of Re neurons allowing us to better consider the role of the Re in cognitive processes.
Abstract
The nucleus reuniens (Re) is the largest of the midline thalamic nuclei. We have performed a detailed neurophysiological characterization of neurons in the rostral Re of brain slices prepared from adult male mice. At resting potential (−63.7 ± 0.6 mV), ∼90% of Re neurons fired action potentials, typically continuously at ∼8 Hz. Although Re neurons experience a significant spontaneous barrage of fast, amino‐acid‐mediate synaptic transmission, this was not predominantly responsible for spontaneous spiking because firing persisted in the presence of glutamate and GABA receptor antagonists. With resting potential preset to −80 mV, −20 pA current injections revealed a mean input resistance of 615 MΩ and a mean time constant of 38 ms. Following cessation of this stimulus, a significant rebound potential was seen that was sometimes sufficiently large to trigger a short burst of very high frequency (100–300 Hz) firing. In most cells, short (2 ms), strong (2 nA) current injections elicited a single spike followed by a large afterdepolarizing potential which, when suprathreshold, generated high‐frequency spiking. Similarly, in the majority of cells preset at −80 mV, 500 ms depolarizing current injections to cells led to a brief initial burst of very high‐frequency firing, although this was lost when cells were preset at −72 mV. Biophysical and pharmacological experiments indicate a prominent role for T‐type Ca2+ channels in the high‐frequency bursting of Re neurons. Finally, we describe a novel form of activity‐dependent intrinsic plasticity that persistently eliminates the burst firing potential of Re neurons.

Key points
5‐HT is a neuromodulator released from carotid body (CB) chemoreceptor (type I) cells and facilitates the sensory discharge following chronic intermittent hypoxia (CIH).
In the present study, we show that, in addition to type I cells, adjacent glial‐like type II cells express functional, ketanserin‐sensitive 5‐HT2 receptors, and their stimulation increases cytoplasmic Ca2+ derived from intracellular stores.
In type II cells, 5‐HT activated a ketanserin‐sensitive inward current (I5‐HT) that was similar to that (IUTP) activated by the P2Y2R agonist, UTP.
As previously shown for IUTP, I5‐HT was inhibited by BAPTA‐AM and carbenoxolone (5 μm), a putative blocker of ATP‐permeable pannexin (Panx)‐1 channels; IUTP was reversibly inhibited by the specific Panx‐1 mimetic peptide channel blocker, 10Panx peptide.
Paracrine stimulation of type II cells by 5‐HT, leading to ATP release via Panx‐1 channels, may contribute to CB excitability, especially in pathophysiological conditions associated with CIH (e.g. obstructive sleep apnoea).
Abstract
Carotid body (CB) chemoreceptor (type I) cells can synthesize and release 5‐HT and increased autocrine–paracrine 5‐HT2 receptor signalling contributes to sensory long‐term facilitation during chronic intermittent hypoxia (CIH). However, recent studies suggest that adjacent glial‐like type II cells can respond to CB paracrine signals by elevating intracellular calcium (Δ[Ca2+]i) and activating carbenoxolone‐sensitive, ATP‐permeable, pannexin (Panx)‐1‐like channels. In the present study, using dissociated rat CB cultures, we found that 5‐HT induced Δ[Ca2+]i responses in a subpopulation of type I cells, as well as in most (∼67%) type II cells identified by their sensitivity to the P2Y2 receptor agonist, UTP. The 5‐HT‐induced Ca2+ response in type II cells was dose‐dependent (EC50 ∼183 nm) and largely inhibited by the 5‐HT2A receptor blocker, ketanserin (1 μm), and also arose mainly from intracellular stores. 5‐HT also activated an inward current (I5‐HT) in type II cells (EC50 ∼200 nm) that was reversibly inhibited by ketanserin (1–10 nm), the Ca2+ chelator BAPTA‐AM (5 μm), and low concentrations of carbenoxolone (5 μm), a putative Panx‐1 channel blocker. I5‐HT reversed direction at approximately −11 mV and was indistinguishable from the UTP‐activated current (IUTP). Consistent with a role for Panx‐1 channels, IUTP was reversibly inhibited by the specific Panx‐1 mimetic peptide blocker 10Panx (100 μm), although not by its scrambled control peptide (scPanx). Because ATP is an excitatory CB neurotransmitter, it is possible that the contribution of enhanced 5‐HT signalling to the increased sensory discharge during CIH may occur, in part, by a boosting of ATP release from type II cells via Panx‐1 channels.

Key points
The brainstem dorsal column nuclei (DCN) process sensory information arising from the body before it reaches the brain and becomes conscious.
Despite significant investigations into sensory coding in peripheral nerves and the somatosensory cortex, little is known about how sensory information arising from the periphery is represented in the DCN.
Following stimulation of hind‐limb nerves, we mapped and characterised the evoked electrical signatures across the DCN surface.
We show that evoked responses recorded from the DCN surface are highly reproducible and are unique to nerves carrying specific sensory information.
Abstract
The brainstem dorsal column nuclei (DCN) play a role in early processing of somatosensory information arising from a variety of functionally distinct peripheral structures, before being transmitted to the cortex via the thalamus. To improve our understanding of how sensory information is represented by the DCN, we characterised and mapped low‐ (<200 Hz) and high‐frequency (550–3300 Hz) components of nerve‐evoked DCN surface potentials. DCN surface potentials were evoked by electrical stimulation of the left and right nerves innervating cutaneous structures (sural nerve), or a mix of cutaneous and deep structures (peroneal nerve), in 8‐week‐old urethane‐anaesthetised male Wistar rats. Peroneal nerve‐evoked DCN responses demonstrated low‐frequency events with significantly longer durations, more high‐frequency events and larger magnitudes compared to responses evoked from sural nerve stimulation. Hotspots of low‐ and high‐frequency DCN activity were found ipsilateral to stimulated nerves but were not symmetrically organised. In conclusion, we find that sensory inputs from peripheral nerves evoke unique and characteristic DCN activity patterns that are highly reproducible both within and across animals.

Key points
Our study contributes to the characterization of muscle loss and weakness processes induced by a sedentary life style, chronic hypoactivity, clinical bed rest, immobilization and microgravity.
This study, by bringing together integrated and cellular evaluation of muscle structure and function, identifies the early functional markers and biomarkers of muscle deconditioning.
Three days of muscle disuse in healthy adult subjects is sufficient to significantly decrease muscle mass, tone and force, and to induce changes in function relating to a weakness in aerobic metabolism and muscle fibre denervation.
The outcomes of this study should be considered in the development of an early muscle loss prevention programme and/or the development of pre‐conditioning programmes required before clinical bed rest, immobilization and spaceflight travel.
Abstract
Microgravity and hypoactivity are associated with skeletal muscle deconditioning. The decrease of muscle mass follows an exponential decay, with major changes in the first days. The purpose of the study was to dissect out the effects of a short‐term 3‐day dry immersion (DI) on human quadriceps muscle function and structure. The DI model, by suppressing all support zones, accurately reproduces the effects of microgravity. Twelve healthy volunteers (32 ± 5 years) completed 3 days of DI. Muscle function was investigated through maximal voluntary contraction (MVC) tests and muscle viscoelasticity. Structural experiments were performed using MRI analysis and invasive experiments on muscle fibres. Our results indicated a significant 9.1% decrease of the normalized MVC constant (P = 0.048). Contraction and relaxation modelization kinetics reported modifications related to torque generation (kACT = −29%; P = 0.014) and to the relaxation phase (kREL = +34%; P = 0.040) after 3 days of DI. Muscle viscoelasticity was also altered. From day one, rectus femoris stiffness and tone decreased by, respectively, 7.3% (P = 0.002) and 10.2% (P = 0.002), and rectus femoris elasticity decreased by 31.5% (P = 0.004) after 3 days of DI. At the cellular level, 3 days of DI translated into a significant atrophy of type I muscle fibres (−10.6 ± 12.1%, P = 0.027) and an increased proportion of hybrid, type I/IIX fibre co‐expression. Finally, we report an increase (6‐fold; P = 0.002) in NCAM+ muscle fibres, showing an early denervation process. This study is the first to report experiments performed in Europe investigating human short‐term DI‐induced muscle adaptations, and contributes to deciphering the early changes and biomarkers of skeletal muscle deconditioning.

Key points
Direct current stimulation (DCS) polarity specifically modulates synaptic efficacy during a continuous train of presynaptic inputs, despite synaptic depression.
DCS polarizes afferent axons and postsynaptic neurons, boosting cooperativity between synaptic inputs.
Polarization of afferent neurons in upstream brain regions may modulate activity in the target brain region during transcranial DCS (tDCS).
A statistical theory of coincident activity predicts that the diffuse and weak profile of current flow can be advantageous in enhancing connectivity between co‐active brain regions.
Abstract
Transcranial direct current stimulation (tDCS) produces sustained and diffuse current flow in the brain with effects that are state dependent and outlast stimulation. A mechanistic explanation for tDCS should capture these spatiotemporal features. It remains unclear how sustained DCS affects ongoing synaptic dynamics and how modulation of afferent inputs by diffuse stimulation changes synaptic activity at the target brain region. We tested the effect of acute DCS (10–20 V m−1 for 3–5 s) on synaptic dynamics with constant rate (5–40 Hz) and Poisson‐distributed (4 Hz mean) trains of presynaptic inputs. Across tested frequencies, sustained synaptic activity was modulated by DCS with polarity‐specific effects. Synaptic depression attenuates the sensitivity to DCS from 1.1% per V m−1 to 0.55%. DCS applied during synaptic activity facilitates cumulative neuromodulation, potentially reversing endogenous synaptic depression. We establish these effects are mediated by both postsynaptic membrane polarization and afferent axon fibre polarization, which boosts cooperativity between synaptic inputs. This potentially extends the locus of neuromodulation from the nominal target to afferent brain regions. Based on these results we hypothesized the polarization of afferent neurons in upstream brain regions may modulate activity in the target brain region during tDCS. A multiscale model of transcranial electrical stimulation including a finite element model of brain current flow, numerical simulations of neuronal activity, and a statistical theory of coincident activity predicts that the diffuse and weak profile of current flow can be advantageous. Thus, we propose that specifically because tDCS is diffuse, weak and sustained it can boost connectivity between co‐active brain regions.

Key points
Ageing is associated with an upregulation of mitochondrial dynamics proteins mitofusin 2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49) in human skeletal muscle with the increased abundance of Mfn2 being exclusive to type II muscle fibres.
These changes occur despite a similar content of mitochondria, as measured by COXIV, NDUFA9 and complexes in their native states (Blue Native PAGE).
Following 12 weeks of high‐intensity training (HIT), older adults exhibit a robust increase in mitochondria content, while there is a decline in Mfn2 in type II fibres.
We propose that the upregulation of Mfn2 and MiD49 with age may be a protective mechanism to protect against mitochondrial dysfunction, in particularly in type II skeletal muscle fibres, and that exercise may have a unique protective effect negating the need for an increased turnover of mitochondria.
Abstract
Mitochondrial dynamics proteins are critical for mitochondrial turnover and maintenance of mitochondrial health. High‐intensity interval training (HIT) is a potent training modality shown to upregulate mitochondrial content in young adults but little is known about the effects of HIT on mitochondrial dynamics proteins in older adults. This study investigated the abundance of protein markers for mitochondrial dynamics and mitochondrial content in older adults compared to young adults. It also investigated the adaptability of mitochondria to 12 weeks of HIT in older adults. Both older and younger adults showed a higher abundance of mitochondrial respiratory chain subunits COXIV and NDUFA9 in type I compared with type II fibres, with no difference between the older adults and young groups. In whole muscle homogenates, older adults had higher mitofusin‐2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49) contents compared to the young group. Also, older adults had higher levels of Mfn2 in type II fibres compared with young adults. Following HIT in older adults, MiD49 and Mfn2 levels were not different in whole muscle and Mfn2 content decreased in type II fibres. Increases in citrate synthase activity (55%) and mitochondrial respiratory chain subunits COXIV (37%) and NDUFA9 (48%) and mitochondrial respiratory chain complexes (∼70–100%) were observed in homogenates and/or single fibres. These findings reveal (i) a similar amount of mitochondria in muscle from young and healthy older adults and (ii) a robust increase of mitochondrial content following 12 weeks of HIT exercise in older adults.

Key points
Spontaneous activity of the sensory inner hair cells shapes maturation of the developing ascending (afferent) auditory system before hearing begins.
Just before the onset of hearing, descending (efferent) input from cholinergic neurons originating in the brainstem inhibit inner hair cell spontaneous activity and may further refine maturation.
We show that agonist activation of the group I metabotropic glutamate receptor mGluR1 increases the strength of this efferent inhibition by enhancing the presynaptic release of acetylcholine.
We further show that the endogenous release of glutamate from the inner hair cells may increase the strength of efferent inhibition via the activation of group I metabotropic glutamate receptors.
Thus, before the onset of hearing, metabotropic glutamate signalling establishes a local negative feedback loop that is positioned to regulate inner hair cell excitability and refine maturation of the auditory system.
Abstract
Just before the onset of hearing, the inner hair cells (IHCs) receive inhibitory efferent input from cholinergic medial olivocochlear (MOC) neurons originating in the brainstem. This input may serve a role in the maturation of the ascending (afferent) auditory system by inhibiting spontaneous activity of the IHCs. To investigate the molecular mechanisms regulating these IHC efferent synapses, we combined electrical stimulation of the efferent fibres with patch clamp recordings from the IHCs to measure efferent synaptic strength. By examining evoked responses, we show that activation of metabotropic glutamate receptors (mGluRs) by general and group I‐specific mGluR agonists enhances IHC efferent inhibition. This enhancement is blocked by application of a group I mGluR1‐specific antagonist, indicating that enhancement of IHC efferent inhibition is mediated by group I mGluRs and specifically by mGluR1s. By comparing spontaneous and evoked responses, we show that group I mGluR agonists act presynaptically to increase neurotransmitter release without affecting postsynaptic responsiveness. Moreover, endogenous glutamate released from the IHCs also enhances IHC efferent inhibition via the activation of group I mGluRs. Finally, immunofluorescence analysis indicates that the efferent terminals are sufficiently close to IHC glutamate release sites to allow activation of mGluRs on the efferent terminals by glutamate spillover. Together, these results suggest that glutamate released from the IHCs activates group I mGluRs (mGluR1s), probably present on the efferent terminals, which, in turn, enhances release of acetylcholine and inhibition of the IHCs. Thus, mGluRs establish a local negative feedback loop positioned to regulate IHC activity and maturation of the ascending auditory system in the developing cochlea.

Key points
Dysfunctions in CNS regulation of arterial blood pressure lead to an increase in sympathetic nerve activity that participates in the pathogenesis of hypertension.
The apelin‐apelin receptor system affects arterial blood pressure homeostasis; however, the central mechanisms underlying apelin‐mediated changes in sympathetic nerve activity and blood pressure have not been clarified.
We explored the mechanisms involved in the regulation of [Pyr1]apelin‐13‐mediated cardiovascular control within the rostral ventrolateral medulla (RVLM) using selective receptor antagonists.
We show that [Pyr1]apelin‐13 acts as a modulating neurotransmitter in the normotensive RVLM to affect vascular tone through interaction with the vasopressin V1a receptor but that [Pyr1]apelin‐13‐induced sympathoexcitation is independent of angiotensin II receptor type 1, oxytocin, ionotropic glutamate and GABAA receptors.
Our data confirm a role for the apelin peptide system in cardiovascular regulation at the level of the RVLM and highlight that this system is a possible potential therapeutic target for the treatment of hypertension.
Abstract
Apelin is a ubiquitous peptide that can elevate arterial blood pressure (ABP) yet understanding of the mechanisms involved remain incomplete. Bilateral microinjection of [Pyr1]apelin‐13 into the rostral ventrolateral medulla (RVLM), a major source of sympathoexcitatory neurones, increases ABP and sympathetic nerve activity. We aimed to investigate the potential involvement of neurotransmitter systems through which the apelin pressor response may occur within the RVLM. Adult male Wistar rats were anaesthetized and ABP was monitored via a femoral arterial catheter. Bilateral RVLM microinjection of [Pyr1]apelin‐13 significantly increased ABP (9 ± 1 mmHg) compared to saline (−1 ± 2mmHg; P 0.05). By contrast, the [Pyr1]apelin‐13 induced pressor and sympathoexcitatory effects were abolished by pretreatment of the RVLM with the vasopressin V1a receptor antagonist, SR 49059 (−1 ± 1 mmHg; 1.1 ± 1.1% total power, respectively; P

Key points
Angiotensin II (AngII) is crucial in cardiovascular regulation in perinatal mammalians.
Here we show that AngII increases twitch Ca2+ transients of mouse immature but not mature cardiomyocytes by robustly activating CaV1.2 L‐type Ca2+ channels through a novel signalling pathway involving angiotensin type 1 (AT1) receptors, β‐arrestin2 and casein kinase 2.
A β‐arrestin‐biased AT1 receptor agonist, TRV027, was as effective as AngII in activating L‐type Ca2+ channels.
Our results help understand the molecular mechanism by which AngII regulates the perinatal circulation and also suggest that β‐arrestin‐biased AT1 receptor agonists may be valuable therapeutics for paediatric heart failure.
Abstract
Angiotensin II (AngII), the main effector peptide of the renin–angiotensin system, plays important roles in cardiovascular regulation in the perinatal period. Despite the well‐known stimulatory effect of AngII on vascular contraction, little is known about regulation of contraction of the immature heart by AngII. Here we found that AngII significantly increased the peak amplitude of twitch Ca2+ transients by robustly activating L‐type CaV1.2 Ca2+ (CaV1.2) channels in mouse immature but not mature cardiomyocytes. This response to AngII was mediated by AT1 receptors and β‐arrestin2. A β‐arrestin‐biased AT1 receptor agonist was as effective as AngII in activating CaV1.2 channels. Src‐family tyrosine kinases (SFKs) and casein kinase 2α’β (CK2α’β) were sequentially activated when AngII activated CaV1.2 channels. A cyclin‐dependent kinase inhibitor, p27Kip1 (p27), inhibited CK2α’β, and AngII removed this inhibitory effect through phosphorylating tyrosine 88 of p27 via SFKs in cardiomyocytes. In a human embryonic kidney cell line, tsA201 cells, overexpression of CK2α’β but not c‐Src directly activated recombinant CaV1.2 channels composed of C‐terminally truncated α1C, the distal C‐terminus of α1C, β2C and α2δ1 subunits, by phosphorylating threonine 1704 located at the interface between the proximal and the distal C‐terminus of CaV1.2α1C subunits. Co‐immunoprecipitation revealed that CaV1.2 channels, CK2α’β and p27 formed a macromolecular complex. Therefore, stimulation of AT1 receptors by AngII activates CaV1.2 channels through β‐arrestin2 and CK2α’β, thereby probably exerting a positive inotropic effect in the immature heart. Our results also indicated that β‐arrestin‐biased AT1 receptor agonists may be used as valuable therapeutics for paediatric heart failure in the future.

Key points
Chronic alcohol consumption causes a spectrum of liver diseases, but the pathogenic mechanisms driving the onset and progression of disease are not clearly defined.
We show that chronic alcohol feeding sensitizes rat hepatocytes to Ca2+‐mobilizing hormones resulting in a leftward shift in the concentration–response relationship and the transition from oscillatory to more sustained and prolonged Ca2+ increases.
Our data demonstrate that alcohol‐dependent adaptation in the Ca2+ signalling pathway occurs at the level of hormone‐induced inositol 1,4,5 trisphosphate (IP3) production and does not involve changes in the sensitivity of the IP3 receptor or size of internal Ca2+ stores.
We suggest that prolonged and aberrant hormone‐evoked Ca2+ increases may stimulate the production of mitochondrial reactive oxygen species and contribute to alcohol‐induced hepatocyte injury.
Abstract
‘Adaptive’ responses of the liver to chronic alcohol consumption may underlie the development of cell and tissue injury. Alcohol administration can perturb multiple signalling pathways including phosphoinositide‐dependent cytosolic calcium ([Ca2+]i) increases, which can adversely affect mitochondrial Ca2+ levels, reactive oxygen species production and energy metabolism. Our data indicate that chronic alcohol feeding induces a leftward shift in the dose–response for Ca2+‐mobilizing hormones resulting in more sustained and prolonged [Ca2+]i increases in both cultured hepatocytes and hepatocytes within the intact perfused liver. Ca2+ increases were initiated at lower hormone concentrations, and intercellular calcium wave propagation rates were faster in alcoholics compared to controls. Acute alcohol treatment (25 mm) completely inhibited hormone‐induced calcium increases in control livers, but not after chronic alcohol‐feeding, suggesting desensitization to the inhibitory actions of ethanol. Hormone‐induced inositol 1,4,5 trisphosphate (IP3) accumulation and phospholipase C (PLC) activity were significantly potentiated in hepatocytes from alcohol‐fed rats compared to controls. Removal of extracellular calcium, or chelation of intracellular calcium did not normalize the differences in hormone‐stimulated PLC activity, indicating calcium‐dependent PLCs are not upregulated by alcohol. We propose that the liver ‘adapts’ to chronic alcohol exposure by increasing hormone‐dependent IP3 formation, leading to aberrant calcium increases, which may contribute to hepatocyte injury.

Key points
Visual input to the suprachiasmatic nucleus circadian clock is critical for animals to adapt their physiology and behaviour in line with the solar day.
In addition to direct retinal projections, the clock receives input from the visual thalamus, although the role of this geniculohypothalamic pathway in circadian photoreception is poorly understood.
In the present study, we develop a novel brain slice preparation that preserves the geniculohypothalamic pathway to show that GABAergic thalamic neurons inhibit retinally‐driven activity in the central clock in a circadian time‐dependent manner.
We also show that in vivo manipulation of thalamic signalling adjusts specific features of the hypothalamic light response, indicating that the geniculohypothalamic pathway is primarily activated by crossed retinal inputs.
Our data provide a mechanism by which geniculohypothalamic signals can adjust the magnitude of circadian and more acute hypothalamic light responses according to time‐of‐day and establish an important new model for future investigations of the circadian visual system.
Abstract
Sensory input to the master mammalian circadian clock, the suprachiasmatic nucleus (SCN), is vital in allowing animals to optimize physiology and behaviour alongside daily changes in the environment. Retinal inputs encoding changes in external illumination provide the principle source of such information. The SCN also receives input from other retinorecipient brain regions, primarily via the geniculohypothalamic tract (GHT), although the contribution of these indirect projections to circadian photoreception is currently poorly understood. To address this deficit, in the present study, we established an in vitro mouse brain slice preparation that retains connectivity across the extended circadian system. Using multi‐electrode recordings, we first confirm that this preparation retains intact optic projections to the SCN, thalamus and pretectum and a functional GHT. We next show that optogenetic activation of GHT neurons selectively suppresses SCN responses to retinal input, and also that this effect exhibits a pronounced day/night variation and involves a GABAergic mechanism. This inhibitory action was not associated with overt circadian rhythmicity in GHT output, indicating modulation at the SCN level. Finally, we use in vivo electrophysiological recordings alongside pharmacological inactivation or optogenetic excitation to show that GHT signalling actively modulates specific features of the SCN light response, indicating that GHT cells are primarily activated by crossed retinal projections. Taken together, our data establish a new model for studying network communication in the extended circadian system and provide novel insight into the roles of GHT‐signalling, revealing a mechanism by which thalamic activity can help gate retinal input to the SCN according to time of day.

Key points
The integrity of the baroreflex control of sympathetic activity in heart failure (HF) remains under debate.
We proposed the use of the sequence method to assess the baroreflex control of renal sympathetic nerve activity (RSNA).
The sequence method assesses the spontaneous arterial pressure (AP) fluctuations and their related changes in heart rate (or other efferent responses), providing the sensitivity and the effectiveness of the baroreflex. Effectiveness refers to the fraction of spontaneous AP changes that elicits baroreflex‐mediated variations in the efferent response.
Using three different approaches, we showed that the baroreflex sensitivity between AP and RSNA is not altered in early HF rats. However, the sequence method provided evidence that the effectiveness of baroreflex in changing RSNA in response to AP changes is markedly decreased in HF.
The results help us better understand the baroreflex control of the sympathetic nerve activity.
Abstract
In heart failure (HF), the reflex control of the heart rate is known to be markedly impaired; however, the baroreceptor control of the sympathetic drive remains under debate. Applying the sequence method to a series of arterial pressure (AP) and renal sympathetic nerve activity (RSNA), we demonstrated a clear dysfunction in the baroreflex control of sympathetic activity in rats with early HF. We analysed the baroreflex control of the sympathetic drive using three different approaches: AP vs. RSNA curve, cross‐spectral analysis and sequence method between AP and RSNA. The sequence method also provides the baroreflex effectiveness index (BEI), which represents the percentage of AP ramps that actually produce a reflex response. The methods were applied to control rats and rats with HF induced by myocardial infarction. None of the methods employed to assess the sympathetic baroreflex gain were able to detect any differences between the control and the HF group. However, rats with HF exhibited a lower BEI compared to the controls. Moreover, an optimum delay of 1 beat was observed, i.e. 1 beat is required for the RSNA to respond after AP changing, which corroborates with the findings related to the timing between these two variables. For delay 1, the BEI of the controls was 0.45 ± 0.03, whereas the BEI of rats with HF was 0.29 ± 0.09 (P

Key points
In the Western world, obesogenic diets containing high fat and high sugar (HFHS) are commonly consumed during pregnancy, although their effects on the metabolism of the mother, in relation to feto‐placental glucose utilization and growth, are unknown.
In the present study, the consumption of an obesogenic HFHS diet compromised maternal glucose tolerance and insulin sensitivity in late pregnancy in association with dysregulated lipid and glucose handling by the dam.
These maternal metabolic changes induced by HFHS feeding were related to altered feto‐placental glucose metabolism and growth.
A HFHS diet during pregnancy therefore causes maternal metabolic dysfunction with consequences for maternal nutrient allocation for fetal growth.
These findings have implications for the health of women and their infants, who consume obesogenic diets during pregnancy.
Abstract
In the Western world, obesogenic diets containing high fat and high sugar (HFHS) are commonly consumed during pregnancy. However, the impacts of a HFHS diet during pregnancy on maternal insulin sensitivity and signalling in relation to feto‐placental growth and glucose utilization are unknown. The present study examined the effects of a HFHS diet during mouse pregnancy on maternal glucose tolerance and insulin resistance, as well as, on feto‐placental glucose metabolism. Female mice were fed a control or HFHS diet from day (D) 1 of pregnancy (term = D20.5). At D16 or D19, dams were assessed for body composition, metabolite and hormone concentrations, tissue abundance of growth and metabolic signalling pathways, glucose tolerance and utilization and insulin sensitivity. HFHS feeding perturbed maternal insulin sensitivity in late pregnancy; hepatic insulin sensitivity was higher, whereas sensitivity of the skeletal muscle and white adipose tissue was lower in HFHS than control dams. These changes were accompanied by increased adiposity and reduced glucose production and glucose tolerance of HFHS dams. The HFHS diet also disturbed the hormone and metabolite milieu and altered expression of growth and metabolic signalling pathways in maternal tissues. Furthermore, HFHS feeding was associated with impaired feto‐placental glucose metabolism and growth. A HFHS diet during pregnancy therefore causes maternal metabolic dysfunction with consequences for maternal nutrient allocation for fetal growth. These findings have implications for the health of women and their infants, who consume HFHS diets during pregnancy.

Folate is a water‐soluble B vitamin that is essential for cellular methylation reactions and DNA synthesis and repair. Low maternal folate levels in pregnancy are associated with fetal growth restriction, however the underlying mechanisms are poorly understood. Mechanistic target of rapamycin (mTOR) links nutrient availability to cell growth and function by regulating gene expression and protein translation. Here we show that mTOR functions as a folate sensor in primary human trophoblast (PHT) cells. Folate deficiency in PHT cells caused inhibition of mTOR signalling and decreased the activity of key amino acid transporters. Folate sensing by mTOR in PHT cells involves both mTOR Complex 1 and 2 and requires the proton‐coupled folate transporter (PCFT, SLC46A1). The involvement of PCFT in mTOR folate sensing is not dependent on its function as a plasma membrane folate transporter. Increasing levels of homocysteine had no effect on PHT mTOR signalling, suggesting that mTOR senses low folate rather than high homocysteine. In addition, we demonstrate that maternal serum folate is positively correlated to placental mTORC1 and mTORC2 signalling activity in human pregnancy. We have identified a previously unknown molecular link between folate availability and cell function involving PCFT and mTOR signalling. We propose that mTOR folate sensing in trophoblast cells matches placental nutrient transport, and therefore fetal growth, to folate availability. These findings may have implications for our understanding of how altered folate availability causes human diseases such as fetal growth restriction, fetal malformations and cancer.
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Key points
There are serotonergic projections to both the main (MOB) and the accessory olfactory bulb (AOB).
Current‐clamp experiments demonstrate that serotonergic afferents are largely excitatory for mitral cells (MCs) in the MOB where 5‐HT2A receptors mediate a direct excitatory action.
Serotonergic afferents are predominately inhibitory for MCs in the AOB. There are two types of inhibition: indirect inhibition mediated through the 5‐HT2 receptors on GABAergic interneurons and direct inhibition via the 5‐HT1 receptors on MCs.
Differential 5‐HT neuromodulation of MCs across the MOB and AOB could contribute to select behaviours such as olfactory learning or aggression.
Abstract
Mitral cells (MCs) contained in the main (MOB) and accessory (AOB) olfactory bulb have distinct intrinsic membrane properties but the extent of neuromodulation across the two systems has not been widely explored. Herein, we investigated a widely distributed CNS modulator, serotonin (5‐HT), for its ability to modulate the biophysical properties of MCs across the MOB and AOB, using an in vitro, brain slice approach in postnatal 15–30 day mice. In the MOB, 5‐HT elicited three types of responses in 93% of 180 cells tested. Cells were either directly excited (70%), inhibited (10%) or showed a mixed response (13%)– first inhibition followed by excitation. In the AOB, 82% of 148 cells were inhibited with 18% of cells showing no response. Albeit located in parallel partitions of the olfactory system, 5‐HT largely elicited MC excitation in the MOB while it evoked two different kinetic rates of MC inhibition in the AOB. Using a combination of pharmacological agents, we found that the MC excitatory responses in the MOB were mediated by 5‐HT2A receptors through a direct activation. In comparison, 5‐HT‐evoked inhibitory responses in the AOB arose due to a polysynaptic, slow‐onset inhibition attributed to 5‐HT2 receptor activation exciting GABAergic interneurons. The second type of inhibition had a rapid onset as a result of direct inhibition mediated by the 5‐HT1 class of receptors. The distinct serotonergic modulation of MCs between the MOB and AOB could provide a molecular basis for differential chemosensory behaviours driven by the brainstem raphe nuclei into these parallel systems.

Key points
We constructed a comprehensive mathematical model of the spontaneous electrical activity of a human sinoatrial node (SAN) pacemaker cell, starting from the recent Severi–DiFrancesco model of rabbit SAN cells.
Our model is based on electrophysiological data from isolated human SAN pacemaker cells and closely matches the action potentials and calcium transient that were recorded experimentally.
Simulated ion channelopathies explain the clinically observed changes in heart rate in corresponding mutation carriers, providing an independent qualitative validation of the model.
The model shows that the modulatory role of the ‘funny current’ (If) in the pacing rate of human SAN pacemaker cells is highly similar to that of rabbit SAN cells, despite its considerably lower amplitude.
The model may prove useful in the design of experiments and the development of heart‐rate modulating drugs.
Abstract
The sinoatrial node (SAN) is the normal pacemaker of the mammalian heart. Over several decades, a large amount of data on the ionic mechanisms underlying the spontaneous electrical activity of SAN pacemaker cells has been obtained, mostly in experiments on single cells isolated from rabbit SAN. This wealth of data has allowed the development of mathematical models of the electrical activity of rabbit SAN pacemaker cells. The present study aimed to construct a comprehensive model of the electrical activity of a human SAN pacemaker cell using recently obtained electrophysiological data from human SAN pacemaker cells. We based our model on the recent Severi–DiFrancesco model of a rabbit SAN pacemaker cell. The action potential and calcium transient of the resulting model are close to the experimentally recorded values. The model has a much smaller ‘funny current’ (If) than do rabbit cells, although its modulatory role is highly similar. Changes in pacing rate upon the implementation of mutations associated with sinus node dysfunction agree with the clinical observations. This agreement holds for both loss‐of‐function and gain‐of‐function mutations in the HCN4, SCN5A and KCNQ1 genes, underlying ion channelopathies in If, fast sodium current and slow delayed rectifier potassium current, respectively. We conclude that our human SAN cell model can be a useful tool in the design of experiments and the development of drugs that aim to modulate heart rate.

Recent results have demonstrated modification of electrical synapse strength by varied forms of neuronal activity. However, the mechanisms underlying plasticity induction in central mammalian neurons are unclear. Here we show that the two established inductors of plasticity at electrical synapses in the thalamic reticular nucleus – paired burst spiking in coupled neurons, and mGluR‐dependent tetanization of synaptic input – are separate pathways that converge at a common downstream endpoint. Using occlusion experiments and pharmacology in patched pairs of coupled neurons in vitro, we show that burst‐induced depression depends on calcium entry via voltage‐gated channels, is blocked by BAPTA chelation, and recruits intracellular calcium release on its way to activation of phosphatase activity. In contrast, mGluR‐dependent plasticity is independent of calcium entry or calcium dynamics. Together, these results show that the spiking‐initiated mechanisms underlying electrical synapse plasticity are similar to those that induce plasticity at chemical synapses, and offer the possibility that calcium‐regulated mechanisms may also lead to alternate outcomes, such as potentiation. Because these mechanistic elements are widely found in mature neurons, we expect them to apply broadly to electrical synapses across the brain, acting as the crucial link between neuronal activity and electrical synapse strength.
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Key points
Exercise training effectively improves vascular and skeletal muscle function; however, these effects of training may be blunted in postmenopausal women as a result of the loss of oestrogens.
Accordingly, the capacity to deliver oxygen to the active muscles may also be impaired in postmenopausal women.
In both premenopausal and recent postmenopausal women, exercise training was shown to improve leg vascular and skeletal muscle mitochondrial function. Interestingly, these effects were more pronounced in postmenopausal women.
Skeletal muscle oxygen supply and utilization were similar in the two groups of women.
These findings suggest that the early postmenopausal phase is associated with an enhanced capacity of the leg vasculature and skeletal muscle mitochondria to adapt to exercise training and that the ability to deliver oxygen to match the demand of the active muscles is preserved in the early phase following the menopausal transition.
Abstract
Exercise training leads to favourable adaptations within skeletal muscle; however, this effect of exercise training may be blunted in postmenopausal women as a result of the loss of oestrogens. Furthermore, postmenopausal women may have an impaired vascular response to acute exercise. We examined the haemodynamic response to acute exercise in matched pre‐ and postmenopausal women before and after 12 weeks of aerobic high intensity exercise training. Twenty premenopausal and 16 early postmenopausal (mean ± SEM: 3.1 ± 0.5 years after final menstrual period) women only separated by 4 years of age (mean ± SEM: 50 ± 0 years vs. 54 ± 1 years) were included. Before training, leg blood flow, O2 delivery, O2 uptake and lactate release during knee‐extensor exercise were similar in pre‐ and postmenopausal women. Exercise training reduced (P

Contraction of muscle evokes the exercise pressor reflex (EPR), which is expressed partly by increases in heart rate and arterial pressure. Patients with peripheral artery disease (PAD) show an exaggerated EPR, sometimes report pain when walking and are at risk for cardiac arrthymias. Previous research suggested that reactive oxygen species (ROS) mediate the exaggerated EPR associated with PAD. To examine the effects of ROS on the EPR, we infused a superoxide scavenger, tiron, into the superficial epigastric artery of decerebrated rats. In some, we simulated PAD by ligating a femoral artery for 72 h before the experiment. The peak EPR in “ligated” rats during saline infusion averaged 31 ± 4 mmHg, whereas the peak EPR in these rats during tiron infusion averaged 13 ± 2 mmHg (n = 12; P

Key points
[Ca2+]i enhanced rabbit ventricular slowly activating delayed rectifier K+ current (IKs) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol.
Rabbit ventricular rapidly activating delayed rectifier K+ current (IKr) amplitude and voltage dependence were unaffected by high [Ca2+]i.
When measuring or simulating IKs during an action potential, IKs was not different during a physiological Ca2+ transient or when [Ca2+]i was buffered to 500 nm.
Abstract
The slowly activating delayed rectifier K+ current (IKs) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca2+ ([Ca2+]i) and β‐adrenergic receptor (β‐AR) stimulation modulate IKs amplitude and kinetics, but details of these important IKs regulators and their interaction are limited. We assessed the [Ca2+]i dependence of IKs in steady‐state conditions and with dynamically changing membrane potential and [Ca2+]i during an AP. IKs was recorded from freshly isolated rabbit ventricular myocytes using whole‐cell patch clamp. With intracellular pipette solutions that controlled free [Ca2+]i, we found that raising [Ca2+]i from 100 to 600 nm produced similar increases in IKs as did β‐AR activation, and the effects appeared additive. Both β‐AR activation and high [Ca2+]i increased maximally activated tail IKs, negatively shifted the voltage dependence of activation, and slowed deactivation kinetics. These data informed changes in our well‐established mathematical model of the rabbit myocyte. In both AP‐clamp experiments and simulations, IKs recorded during a normal physiological Ca2+ transient was similar to IKs measured with [Ca2+]i clamped at 500–600 nm. Thus, our study provides novel quantitative data as to how physiological [Ca2+]i regulates IKs amplitude and kinetics during the normal rabbit AP. Our results suggest that micromolar [Ca2+]i, in the submembrane or junctional cleft space, is not required to maximize [Ca2+]i‐dependent IKs activation during normal Ca2+ transients.
[Ca2+]i enhanced rabbit ventricular slowly activating delayed rectifier K+ current (IKs) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol.
Rabbit ventricular rapidly activating delayed rectifier K+ current (IKr) amplitude and voltage dependence were unaffected by high [Ca2+]i.
When measuring or simulating IKs during an action potential, IKs was not different during a physiological Ca2+ transient or when [Ca2+]i was buffered to 500 nm.
Membrane topology of a Kv7.1 α‐subunit and regulatory proteins.

Key points
Vagal sensory inputs transmit information from the viscera to brainstem neurones located in the nucleus tractus solitarii to set physiological parameters. These excitatory synapses exhibit a CB1 endocannabinoid‐induced long‐term depression (LTD) triggered by vagal fibre stimulation.
We investigated the impact of nutritional status on long‐term changes in this long‐term synaptic plasticity.
Food deprivation prevents LTD induction by disrupting CB1 receptor signalling. Short‐term refeeding restores the capacity of vagal synapses to express LTD.
Ghrelin and cholecystokinin, respectively released during fasting and refeeding, play a key role in the control of LTD via the activation of energy sensing pathways such as AMPK and the mTOR and ERK pathways.
Abstract
Communication form the viscera to the brain is essential to set physiological homoeostatic parameters but also to drive more complex behaviours such as mood, memory and emotional states. Here we investigated the impact of the nutritional status on long‐term changes in excitatory synaptic transmission in the nucleus tractus solitarii, a neural hub integrating visceral signals. These excitatory synapses exhibit a CB1 endocannabinoid (eCB)‐induced long‐term depression (LTD) triggered by vagal fibre stimulation. Since eCB signalling is known to be an important component of homoeostatic regulation of the body and is regulated during various stressful conditions, we tested the hypothesis that food deprivation alters eCB signalling in central visceral afferent fibres. Food deprivation prevents eCB‐LTD induction due to the absence of eCB signalling. This loss was reversed by blockade of ghrelin receptors. Activation of the cellular fuel sensor AMP‐activated protein kinase or inhibition of the mechanistic target of rapamycin pathway abolished eCB‐LTD in free‐fed rats. Signals associated with energy surfeit, such as short‐term refeeding, restore eCB‐LTD induction, which in turn requires activation of cholecystokinin receptors and the extracellular signal‐regulated kinase pathway. These data suggest a tight link between eCB‐LTD in the NTS and nutritional status and shed light on the key role of eCB in the integration of visceral information.

Key points
Hypercalcaemia can occur under various pathological conditions, such as primary hyperparathyroidism, malignancy or granulomatosis, and it induces natriuresis and polyuria in various species via an unknown mechanism.
A previous study demonstrated that hypercalcaemia induced by vitamin D in rats increased endothelin (ET)‐1 expression in the distal nephron, which suggests the involvement of the ET system in hypercalcaemia‐induced effects.
In the present study, we demonstrate that, during vitamin D‐induced hypercalcaemia, the activation of ET system by increased ET‐1 is responsible for natriuresis but not for polyuria.
Vitamin D‐treated hypercalcaemic mice showed a blunted response to amiloride, suggesting that epithelial sodium channel function is inhibited.
We have identified an original pathway that specifically mediates the effects of vitamin D‐induced hypercalcaemia on sodium handling in the distal nephron without affecting water handling.
Abstract
Acute hypercalcaemia increases urinary sodium and water excretion; however, the underlying molecular mechanism remains unclear. Because vitamin D‐induced hypercalcaemia increases the renal expression of endothelin (ET)‐1, we hypothesized that ET‐1 mediates the effects of hypercalcaemia on renal sodium and water handling. Hypercalcaemia was induced in 8‐week‐old, parathyroid hormone‐supplemented, male mice by oral administration of dihydrotachysterol (DHT) for 3 days. DHT‐treated mice became hypercalcaemic and displayed increased urinary water and sodium excretion compared to controls. mRNA levels of ET‐1 and the transcription factors CCAAT‐enhancer binding protein β and δ were specifically increased in the distal convoluted tubule and downstream segments in DHT‐treated mice. To examine the role of the ET system in hypercalcaemia‐induced natriuresis and polyuria, mice were treated with the ET‐1 receptor antagonist macitentan, with or without DHT. Mice treated with both macitentan and DHT displayed hypercalcaemia and polyuria similar to that in mice treated with DHT alone; however, no increase in urinary sodium excretion was observed. To identify the affected sodium transport mechanism, we assessed the response to various diuretics in control and DHT‐treated hypercalcaemic mice. Amiloride, an inhibitor of the epithelial sodium channel (ENaC), increased sodium excretion to a lesser extent in DHT‐treated mice compared to control mice. Mice treated with either macitentan+DHT or macitentan alone had a similar response to amiloride. In summary, vitamin D‐induced hypercalcaemia increases the renal production of ET‐1 and decreases ENaC activity, which is probably responsible for the rise in urinary sodium excretion but not for polyuria.

Key points
During lactation, prolactin promotes milk synthesis and oxytocin stimulates milk ejection.
In virgin rats, prolactin inhibits the activity of oxytocin‐secreting neurones.
We found that prolactin inhibition of oxytocin neurone activity is lost in lactation, and that some oxytocin neurones were excited by prolactin in lactating rats.
The change in prolactin regulation of oxytocin neurone activity was not associated with a change in activation of intracellular signalling pathways known to couple to prolactin receptors.
The change in prolactin regulation of oxytocin neurone activity in lactation might allow coordinated activation of both populations of neurones when required for successful lactation.
Abstract
Secretion of prolactin for milk synthesis and oxytocin for milk secretion is required for successful lactation. In virgin rats, prolactin inhibits oxytocin neurones but this effect would be counterproductive during lactation when secretion of both hormones is required for synthesis and delivery of milk to the newborn. Hence, we determined the effects of intracerebroventricular (i.c.v.) prolactin on oxytocin neurones in urethane‐anaesthetised virgin, pregnant and lactating rats. Prolactin (2 μg) consistently inhibited oxytocin neurones in virgin and pregnant rats (by 1.9 ± 0.4 and 1.8 ± 0.5 spikes s−1, respectively), but not in lactating rats; indeed, prolactin excited six of 27 oxytocin neurones by >1 spike s−1 in lactating rats but excited none in virgin or pregnant rats (χ22 = 7.2, P = 0.03). Vasopressin neurones were unaffected by prolactin (2 μg) in virgin rats but were inhibited by 1.1 ± 0.2 spikes s−1 in lactating rats. Immunohistochemistry showed that i.c.v. prolactin increased oxytocin expression in virgin and lactating rats and increased signal transducer and activator of transcription 5 phosphorylation to a similar extent in oxytocin neurones of virgin and lactating rats. Western blotting showed that i.c.v. prolactin did not affect phosphorylation of extracellular regulated kinase 1 or 2, or of Akt in the supraoptic or paraventricular nuclei of virgin or lactating rats. Hence, prolactin inhibition of oxytocin neurones is lost in lactation, which might allow concurrent elevation of prolactin secretion from the pituitary gland and activation of oxytocin neurones for synthesis and delivery of milk to the newborn.

Neural oscillations in the basal ganglia are well studied yet remain poorly understood. Behavioural correlates of spectral activity are well described, yet a quantitative hypothesis linking time domain dynamics and spectral properties to basal ganglia function has been lacking. We show, for the first time, that a unified description is possible by interpreting previously ignored structure in data describing GPi responses to cortical stimulation. These data were used to expose a pair of distinctive neuronal responses to the stimulation. This observation formed the basis for a new mathematical model of the BG, quantitatively fitted to the data, which describes the dynamics in the data, and is validated against other stimulus protocol experiments. A key new result is that when the model is run using inputs hypothesised to occur during the performance of a motor task, beta and gamma frequency oscillations emerge naturally during static‐force and movement respectively, consistent with experimental local field potentials. This new model predicts that the pallido‐striatum connection has a key role in the generation of beta band activity, and that the gamma band activity associated with motor task performance has its origins in the pallido‐subthalamic feedback loop. The network's functionality as a selection‐mechanism also occurs as an emergent property, and closer fits to the data gave better selection properties. The model provides a coherent framework for the study of spectral, temporal and functional analyses of the BG and therefore lays the foundation for an integrated approach to study BG pathologies such as Parkinson's disease in silico.
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Key points
β‐Adrenergic stimulation enhances Ca2+ entry via L‐type CaV1.2 channels, causing stronger contraction of cardiac muscle cells.
The signalling pathway involves activation of protein kinase A (PKA), but the molecular details of PKA regulation of CaV1.2 remain controversial despite extensive research.
We show that PKA regulation of CaV1.2 can be reconstituted in Xenopus oocytes when the distal C‐terminus (dCT) of the main subunit, α1C, is truncated.
The PKA upregulation of CaV1.2 does not require key factors previously implicated in this mechanism: the clipped dCT, the A kinase‐anchoring protein 15 (AKAP15), the phosphorylation sites S1700, T1704 and S1928, or the β subunit of CaV1.2. The gating element within the initial segment of the N‐terminus of the cardiac isoform of α1C is essential for the PKA effect.
We propose that the regulation described here is one of two or several mechanisms that jointly mediate the PKA regulation of CaV1.2 in the heart.
Abstract
β‐Adrenergic stimulation enhances Ca2+ currents via L‐type, voltage‐gated CaV1.2 channels, strengthening cardiac contraction. The signalling via β‐adrenergic receptors (β‐ARs) involves elevation of cyclic AMP (cAMP) levels and activation of protein kinase A (PKA). However, how PKA affects the channel remains controversial. Recent studies in heterologous systems and genetically engineered mice stress the importance of the post‐translational proteolytic truncation of the distal C‐terminus (dCT) of the main (α1C) subunit. Here, we successfully reconstituted the cAMP/PKA regulation of the dCT‐truncated CaV1.2 in Xenopus oocytes, which previously failed with the non‐truncated α1C. cAMP and the purified catalytic subunit of PKA, PKA‐CS, injected into intact oocytes, enhanced CaV1.2 currents by ∼40% (rabbit α1C) to ∼130% (mouse α1C). PKA blockers were used to confirm specificity and the need for dissociation of the PKA holoenzyme. The regulation persisted in the absence of the clipped dCT (as a separate protein), the A kinase‐anchoring protein AKAP15, and the phosphorylation sites S1700 and T1704, previously proposed as essential for the PKA effect. The CaVβ2b subunit was not involved, as suggested by extensive mutagenesis. Using deletion/chimeric mutagenesis, we have identified the initial segment of the cardiac long‐N‐terminal isoform of α1C as a previously unrecognized essential element involved in PKA regulation. We propose that the observed regulation, that exclusively involves the α1C subunit, is one of several mechanisms underlying the overall PKA action on CaV1.2 in the heart. We hypothesize that PKA is acting on CaV1.2, in part, by affecting a structural ‘scaffold’ comprising the interacting cytosolic N‐ and C‐termini of α1C.

Key points
Regular‐spiking interneurons in the hippocampal stratum oriens exhibit a form of long‐term potentiation of excitatory transmission that is independent of NMDA receptors but requires co‐activation of Ca2+‐permeable AMPA receptors and group I metabotropic glutamate receptors.
We show that T‐type Ca2+ channels are present in such interneurons.
Blockade of T‐type currents prevents the induction of long‐term potentiation, and also interferes with long‐lasting potentiation induced either by postsynaptic trains of action potentials or by pairing postsynaptic hyperpolarization with activation of group I metabotropic receptors.
Several Ca2+ sources thus converge on the induction of NMDA receptor independent synaptic plasticity.
Abstract
NMDA receptor independent long‐term potentiation (LTP) in hippocampal stratum oriens‐alveus (O/A) interneurons requires co‐activation of postsynaptic group I metabotropic glutamate receptors (mGluRs) and Ca2+‐permeable AMPA receptors. The rectification properties of such AMPA receptors contribute to the preferential induction of LTP at hyperpolarized potentials. A persistent increase in excitatory transmission can also be triggered by exogenous activation of group I mGluRs at the same time as the interneuron is hyperpolarized, or by postsynaptic trains of action potentials in the absence of presynaptic stimulation. In the present study, we identify low‐threshold transient (T‐type) channels as a further source of Ca2+ that contributes to synaptic plasticity. T‐type Ca2+ currents were detected in mouse regular‐spiking O/A interneurons. Blocking T‐type currents pharmacologically prevented LTP induced by high‐frequency stimulation of glutamatergic axons, or by application of the group I mGluR agonist dihydroxyphenylglycine, paired with postsynaptic hyperpolarization. T‐type current blockade also prevented synaptic potentiation induced by postsynaptic action potential trains. Several sources of Ca2+ thus converge on NMDA receptor independent LTP induction in O/A interneurons.

Key points
Following substantial bleaching of the visual pigment, the desensitization of the rod photovoltage is not as substantial as the desensitization of the rod outer segment photocurrent.
The block of cation conductances during the internal dialysis of Cs+ further desensitizes the photovoltage thereby eliminating its difference in desensitization with the rod outer segment photocurrent.
Bleached visual pigment produced an acceleration of the rod photovoltage with respect to the outer segment photocurrent, which is eliminated upon internal dialysis of Cs+.
Abstract
A majority of our visual experience occurs during the day when a substantial fraction of the visual pigment in our photoreceptor cells is bleached. Under these conditions it is widely believed that rods are saturated and do not contribute substantially to downstream signalling. However, behavioural experiments on subjects with only rod function reveals that these individuals unexpectedly retain substantial vision in daylight. We sought to understand this discrepancy by characterizing the sensitivity of rod photoresponses following exposure to bright bleaching light. Measurements of the rod outer segment photocurrent in transgenic mice, which have only rod function, revealed the well‐studied reduction in the sensitivity of rod photoresponses following pigment bleaching. However, membrane voltage measurements showed that the desensitization of the photovoltage was considerably less than that of the outer segment photocurrent following equivalent pigment bleaching. This discrepancy was largely eliminated during the blockade of cation channels due to the internal dialysis of Cs+, which increased the bleach‐induced desensitization of the photovoltage and slowed its temporal characteristics. Thus, sensitization of the photovoltage by rod inner segment conductances appears to extend the operating range of rod phototransduction following pigment bleaching.

Key points
Optic nerve axons get less excitable with warming.
F‐fibre latency does not shorten at temperatures above 30°C.
Action potential amplitude falls when the Na+‐pump is blocked, an effect speeded by warming.
Diuretics reduce the rate of action potential fall in the presence of ouabain.
Our data are consistent with electroneutral entry of Na+ occurring in axons and contributing to setting the resting potential.
Abstract
Raising the temperature of optic nerve from room temperature to near physiological has effects on the threshold, refractoriness and superexcitability of the shortest latency (fast, F) nerve fibres, consistent with hyperpolarization. The temperature dependence of peak impulse latency was weakened at temperatures above 30°C suggesting a temperature‐sensitive process that slows impulse propagation. The amplitude of the supramaximal compound action potential gets larger on warming, whereas in the presence of bumetanide and amiloride (blockers of electroneutral Na+ movement), the action potential amplitude consistently falls. This suggests a warming‐induced hyperpolarization that is reduced by blocking electroneutral Na+ movement. In the presence of ouabain, the action potential collapses. This collapse is speeded by warming, and exposure to bumetanide and amiloride slows the temperature‐dependent amplitude decline, consistent with a warming‐induced increase in electroneutral Na+ entry. Blocking electroneutral Na+ movement is predicted to be useful in the treatment of temperature‐dependent symptoms under conditions with reduced safety factor (Uhthoff's phenomenon) and provide a route to neuroprotection.

Key points
Lysophosphatidic acid (LPA) is an itch mediator, but not a pain mediator by a cheek injection model.
Dorsal root ganglion neurons directly respond to LPA depending on transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1).
LPA‐induced itch‐related behaviours are decreased in TRPA1‐knockout (KO), TRPV1KO or TRPA1TRPV1 double KO mice.
TRPA1 and TRPV1 channels are activated by intracellular LPA, but not by extracellular LPA following LPA5 receptor activation with an activity of Ca2+‐independent phospholipase A2 and phospholipase D.
Intracellular LPA interaction sites of TRPA1 are KK672–673 and KR977–978 (K: lysine, R: arginine).
Abstract
Intractable and continuous itch sensations often accompany diseases such as atopic dermatitis, neurogenic lesions, uremia and cholestasis. Lysophosphatidic acid (LPA) is an itch mediator found in cholestatic itch patients and it induces acute itch and pain in experimental rodent models. However, the molecular mechanism by which LPA activates peripheral sensory neurons remains unknown. In this study, we used a cheek injection method in mice to reveal that LPA induced itch‐related behaviours but not pain‐related behaviours. The LPA‐induced itch behaviour and cellular effects were dependent on transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1), which are important for itch signal transduction. We also found that, among the six LPA receptors, the LPA5 receptor had the greatest involvement in itching. Furthermore, we demonstrated that phospholipase D (PLD) plays a critical role downstream of LPA5 and that LPA directly and intracellularly activates TRPA1 and TRPV1. These results suggest a unique mechanism by which cytoplasmic LPA produced de novo could activate TRPA1 and TRPV1. We conclude that LPA‐induced itch is mediated by LPA5, PLD, TRPA1 and TRPV1 signalling, and thus targeting TRPA1, TRPV1 or PLD could be effective for cholestatic itch interventions.

Key points
We found that extracellular Ca2+, but not other divalent cations (Mg2+ and Ba2+) or intracellular Ca2+, is involved in heat‐evoked activation of green anole (ga) TRPA1.
Heat‐evoked activation of chicken (ch) and rat snake (rs) TRPA1 does not depend solely on extracellular Ca2+.
Neutralization of acidic amino acids on the outer surface of TRPA1 by extracellular Ca2+ is important for heat‐evoked large activation of gaTRPA1, chTRPA1 and rsTRPA1.
Abstract
Transient receptor potential ankyrin 1 (TRPA1) is a homotetrameric non‐selective cation‐permeable channel that has six transmembrane domains and cytoplasmic N‐ and C‐termini. The N‐terminus is characterized by an unusually large number of ankyrin repeats. Although the 3‐dimensional structure of human TRPA1 has been determined, and TRPA1 channels from insects to birds are known to be activated by heat stimulus, the mechanism for temperature‐dependent TRPA1 activation is unclear. We previously reported that extracellular Ca2+, but not intracellular Ca2+, plays an important role in heat‐evoked TRPA1 activation in green anole lizards (gaTRPA1). Here we focus on extracellular Ca2+‐dependent heat sensitivity of gaTRPA1 by comparing gaTRPA1 with heat‐activated TRPA1 channels from rat snake (rsTRPA1) and chicken (chTRPA1). In the absence of extracellular Ca2+, rsTRPA1 and chTRPA1 are activated by heat and generate small inward currents. A comparison of extracellular amino acids in TRPA1 identified three negatively charged amino acid residues (glutamate and aspartate) near the outer pore vestibule that are involved in heat‐evoked TRPA1 activation in the presence of extracellular Ca2+. These results suggest that neutralization of acidic amino acids by extracellular Ca2+ is important for heat‐evoked activation of gaTRPA1, chTRPA1, and rsTRPA1, which could clarify mechanisms of heat‐evoked channel activation.

Key points
Sex differences in left ventricular (LV) mechanics occur during acute physiological challenges; however, it is unknown whether sex differences in LV mechanics are fundamentally regulated by differences in adrenergic control.
Using two‐dimensional echocardiography and speckle tracking analysis, this study compared LV mechanics in males and females matched for LV length during post‐exercise ischaemia (PEI) and β1‐adrenergic receptor blockade.
Our data demonstrate that while basal rotation was increased in males, LV twist was not significantly different between the sexes during PEI. In contrast, during β1‐adrenergic receptor blockade, LV apical rotation, twist and untwisting velocity were reduced in males compared to females.
Significant relationships were observed between LV twist and LV internal diameter and sphericity index in females, but not males.
These findings suggest that LV twist mechanics may be more sensitive to alterations in adrenergic stimulation in males, but more highly influenced by ventricular structure and geometry in females.
Abstract
Sex differences in left ventricular (LV) mechanics exist at rest and during acute physiological stress. Differences in cardiac autonomic and adrenergic control may contribute to sex differences in LV mechanics and LV haemodynamics. Accordingly, this study aimed to investigate sex differences in LV mechanics with altered adrenergic stimulation achieved through post‐handgrip‐exercise ischaemia (PEI) and β1‐adrenergic receptor (AR) blockade. Twenty males (23 ± 5 years) and 20 females (22 ± 3 years) were specifically matched for LV length (males: 8.5 ± 0.5 cm, females: 8.2 ± 0.6 cm, P = 0.163), and two‐dimensional speckle‐tracking echocardiography was used to assess LV structure and function at baseline, during PEI and following administration of 5 mg bisoprolol (β1‐AR antagonist). During PEI, LV end‐diastolic volume and stroke volume were increased in both groups (P

Key points
Chloroquine (CQ) stimulates itch nerves and causes intense scratching in mice by activating the G‐protein coupled receptor (GPCR) MrgprA3; it is not known how stimulation of MrgprA3 (or other GPCRs) leads to activation of the itch nerve terminals in the skin, but previous studies have found that transient receptor potential A1 (TRPA1) gene deletion blocks CQ‐induced scratching.
In the present study we used a novel dorsal skin–nerve preparation to evaluate mechanisms underlying CQ‐ and histamine‐induced action potential discharge in itch nerve terminals.
We found that CQ activation of the nerves requires the beta3 isoform of phospholipase C, but TRPA1 or other TRP channel are not required.
Evidence is provided for a role for calcium‐activated chloride channels such as TMEM16a in GPCR‐activation of itch nerve terminals.
The mechanism by which TRP channels participate in pruritogen‐induced scratching may involve sites of action other than the primary afferent terminals.
Abstract
Chloroquine (CQ) and histamine are pruritogens commonly used to study itch in the mouse. A novel skin–nerve preparation was used to evaluate chloroquine (CQ)‐ and histamine‐induced activation of afferent nerves in the dorsal thoracic skin of the mouse. All CQ sensitive nerves were C‐fibres, and were also sensitive to histamine. The response to CQ, but not histamine, was largely absent in mrgpr‐cluster Δ−/− mice, supporting the hypothesis that CQ evokes itch largely via stimulation of MrgprA3 receptors. The CQ‐induced action potential discharge was largely absent in phospholipase Cβ3 knockout animals. The CQ and histamine responses were not influenced by removal of TRPA1, TRPV1, TRPC3 or TRPC6, nor by the TRP channel blocker Ruthenium Red. The bouts of scratching in response to CQ were not different between wild‐type and TRPA1‐deficient mice. A selective inhibitor of the calcium‐activated chloride channel TMEM16A, N‐((4‐methoxy)‐2‐naphthyl)‐5‐nitroanthranilic acid (MONNA), inhibited CQ‐induced action potential discharge at itch nerve terminals and bouts of scratching by about 50%. Although TRPA1 and TRPV1 channels may be involved in the scratching responses to intradermal pruritogens, this is unlikely to be due to an effect at the nerve terminals, where chloride channels may play a more important role.

Key points
Neurodegenerative disorders can exhibit dysfunctional mitochondrial respiratory chain complex IV activity.
Conditional deletion of cytochrome c oxidase, the terminal enzyme in the respiratory electron transport chain of mitochondria, from hippocampal dentate granule cells in mice does not affect low‐frequency dentate to CA3 glutamatergic synaptic transmission.
High‐frequency dentate to CA3 glutamatergic synaptic transmission and feedforward inhibition are significantly attenuated in cytochrome c oxidase‐deficient mice.
Intact presynaptic mitochondrial function is critical for the short‐term dynamics of mossy fibre to CA3 synaptic function.
Abstract
Neurodegenerative disorders are characterized by peripheral and central symptoms including cognitive impairments which have been associated with reduced mitochondrial function, in particular mitochondrial respiratory chain complex IV or cytochrome c oxidase activity. In the present study we conditionally removed a key component of complex IV, protohaem IX farnesyltransferase encoded by the COX10 gene, in granule cells of the adult dentate gyrus. Utilizing whole‐cell patch‐clamp recordings from morphologically identified CA3 pyramidal cells from control and complex IV‐deficient mice, we found that reduced mitochondrial function did not result in overt deficits in basal glutamatergic synaptic transmission at the mossy‐fibre synapse because the amplitude, input–output relationship and 50 ms paired‐pulse facilitation were unchanged following COX10 removal from dentate granule cells. However, trains of stimuli given at high frequency (> 20 Hz) resulted in dramatic reductions in short‐term facilitation and, at the highest frequencies (> 50 Hz), also reduced paired‐pulse facilitation, suggesting a requirement for adequate mitochondrial function to maintain glutamate release during physiologically relevant activity patterns. Interestingly, local inhibition was reduced, suggesting the effect observed was not restricted to synapses with CA3 pyramidal cells via large mossy‐fibre boutons, but rather to all synapses formed by dentate granule cells. Therefore, presynaptic mitochondrial function is critical for the short‐term dynamics of synapse function, which may contribute to the cognitive deficits observed in pathological mitochondrial dysfunction.

Key points
A cerebellar dentate nuclei (DN) contribution to volitional oculomotor control has recently been hypothesized but not fully understood.
Cerebrotendinous xanthomatosis (CTX) is a rare neurometabolic disease typically characterized by DN damage.
In this study, we compared the ocular movement characteristics of two sets of CTX patients, with and without brain MRI evidence of DN involvement, with a set of healthy subjects.
Our results suggest that DN participate in voluntary behaviour, such as the execution of antisaccades, and moreover are involved in controlling the precision of the ocular movement.
The saccadic abnormalities related to DN involvement were independent of global and regional brain atrophy.
Our study confirms the relevant role of DN in voluntary aspects of oculomotion and delineates specific saccadic abnormalities that could be used to detect the involvement of DN in other cerebellar disorders.
Abstract
It is well known that the medial cerebellum controls saccadic speed and accuracy. In contrast, the role of the lateral cerebellum (cerebellar hemispheres and dentate nuclei, DN) is less well understood. Cerebrotendinous xanthomatosis (CTX) is a lipid storage disorder due to mutations in CYP27A1, typically characterized by DN damage. CTX thus provides a unique opportunity to study DN in human oculomotor control. We analysed horizontal and vertical visually guided saccades and horizontal antisaccades of 19 CTX patients. Results were related to the presence/absence of DN involvement and compared with those of healthy subjects. To evaluate the contribution of other areas, abnormal saccadic parameters were compared with global and regional brain volumes. CTX patients executed normally accurate saccades with normal main sequence relationships, indicating that the brainstem and medial cerebellar structures were functionally spared. Patients with CTX executed more frequent multistep saccades and directional errors during the antisaccade task than controls. CTX patients with DN damage showed less precise saccades with longer latencies, and more frequent directional errors, usually not followed by corrections, than either controls or patients without DN involvement. These saccadic abnormalities related to DN involvement but were independent of global and regional brain atrophy. We hypothesize that two different cerebellar networks contribute to the metrics of a movement: the medial cerebellar structures determine accuracy, whereas the lateral cerebellar structures control precision. The lateral cerebellum (hemispheres and DN) also participates in modulating goal directed gaze behaviour, by prioritizing volitional over reflexive movements.

Key points
Cav1.2 channels maintain activity through interactions with calmodulin (CaM). In this study, activities of the Cav1.2 channel (α1C) and of mutant‐derivatives, C‐terminal deleted (α1CΔ) and α1CΔ linked with CaM (α1CΔCaM), were compared in the inside‐out mode.
α1CΔ with CaM, but not without CaM, and α1CΔCaM were active, suggesting that CaM induced channel activity through a dynamic interaction with the channel, even without the distal C‐tail.
ATP induced α1C activity with CaM and enhanced activity of the mutant channels. Okadaic acid mimicked the effect of ATP on the wildtype but not mutant channels.
These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels through their dynamic interactions. ATP effects involve mechanisms both related and unrelated to channel phosphorylation.
CaM‐linked channels are useful tools for investigating Cav1.2 channels in the inside‐out mode; the fast run‐down is prevented by only ATP and the slow run‐down is nearly absent.
Abstract
Calmodulin (CaM) plays a critical role in regulation of Cav1.2 Ca2+ channels. CaM binds to the channel directly, maintaining channel activity and regulating it in a Ca2+‐dependent manner. To explore the molecular mechanisms involved, we compared the activity of the wildtype channel (α1C) and mutant derivatives, C‐terminal deleted (α1C∆) and α1C∆ linked to CaM (α1C∆CaM). These were co‐expressed with β2a and α2δ subunits in HEK293 cells. In the inside‐out mode, α1C and α1C∆ showed minimal open‐probabilities in a basic internal solution (run‐down), whereas α1C∆ with CaM and α1C∆CaM maintained detectable channel activity, confirming that CaM was necessary, but not sufficient, for channel activity. Previously, we reported that ATP was required to maintain channel activity of α1C. Unlike α1C, the mutant channels did not require ATP for activation in the early phase (3–5 min). However, α1C∆ with CaM + ATP and α1C∆CaM with ATP maintained activity, even in the late phase (after 7–9 min). These results suggested that CaM and ATP interacted dynamically with the proximal C‐terminal tail of the channel and, thereby, produced channel activity. In addition, okadaic acid, a protein phosphatase inhibitor, could substitute for the effects of ATP on α1C but not on the mutant channels. These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels, further indicating that ATP has dual effects. One maintains phosphorylation of the channel and the other becomes apparent when the distal carboxyl‐terminal tail is removed.

Key points
Thyroid hormones are important regulators of growth and maturation before birth, although the extent to which their actions are mediated by insulin and the development of pancreatic beta cell mass is unknown.
Hypothyroidism in fetal sheep induced by removal of the thyroid gland caused asymmetric organ growth, increased pancreatic beta cell mass and proliferation, and was associated with increased circulating concentrations of insulin and leptin.
In isolated fetal sheep islets studied in vitro, thyroid hormones inhibited beta cell proliferation in a dose‐dependent manner, while high concentrations of insulin and leptin stimulated proliferation.
The developing pancreatic beta cell is therefore sensitive to thyroid hormone, insulin and leptin before birth, with possible consequences for pancreatic function in fetal and later life.
The findings of this study highlight the importance of thyroid hormones during pregnancy for normal development of the fetal pancreas.
Abstract
Development of pancreatic beta cell mass before birth is essential for normal growth of the fetus and for long‐term control of carbohydrate metabolism in postnatal life. Thyroid hormones are also important regulators of fetal growth, and the present study tested the hypotheses that thyroid hormones promote beta cell proliferation in the fetal ovine pancreatic islets, and that growth retardation in hypothyroid fetal sheep is associated with reductions in pancreatic beta cell mass and circulating insulin concentration in utero. Organ growth and pancreatic islet cell proliferation and mass were examined in sheep fetuses following removal of the thyroid gland in utero. The effects of triiodothyronine (T3), insulin and leptin on beta cell proliferation rates were determined in isolated fetal ovine pancreatic islets in vitro. Hypothyroidism in the sheep fetus resulted in an asymmetric pattern of organ growth, pancreatic beta cell hyperplasia, and elevated plasma insulin and leptin concentrations. In pancreatic islets isolated from intact fetal sheep, beta cell proliferation in vitro was reduced by T3 in a dose‐dependent manner and increased by insulin at high concentrations only. Leptin induced a bimodal response whereby beta cell proliferation was suppressed at the lowest, and increased at the highest, concentrations. Therefore, proliferation of beta cells isolated from the ovine fetal pancreas is sensitive to physiological concentrations of T3, insulin and leptin. Alterations in these hormones may be responsible for the increased beta cell proliferation and mass observed in the hypothyroid sheep fetus and may have consequences for pancreatic function in later life.

Key points
Heart function is critically dependent upon the balance of energy production and utilization. Sarcolemmal ATP‐sensitive potassium channels (KATP channels) in cardiac myocytes adjust contractile function to compensate for the level of available energy.
Understanding the activation of KATP channels in working myocardium during high‐stress situations is crucial to the treatment of cardiovascular disease, especially ischaemic heart disease.
Using a new optical mapping approach, we measured action potentials from the surface of excised contracting rabbit hearts to assess when sarcolemmal KATP channels were activated during physiologically relevant workloads and during gradual reductions in myocardial oxygenation.
We demonstrate that left ventricular pressure is closely linked to KATP channel activation and that KATP channel inhibition with a low concentration of tolbutamide prevents electromechanical decline when oxygen availability is reduced. As a result, KATP channel inhibition probably exacerbates a mismatch between energy demand and energy production when myocardial oxygenation is low.
Abstract
Sarcolemmal ATP‐sensitive potassium channel (KATP channel) activation in isolated cells is generally understood, although the relationship between myocardial oxygenation and KATP activation in excised working rabbit hearts remains unknown. We optically mapped action potentials (APs) in excised rabbit hearts to test the hypothesis that hypoxic changes would be more severe in left ventricular (LV) working hearts (LWHs) than Langendorff (LANG) perfused hearts. We further hypothesized that KATP inhibition would prevent those changes. Optical APs were mapped when measuring LV developed pressure (LVDP), coronary flow rate and oxygen consumption in LANG and LWHs. Hearts were paced to increase workload and perfusate was deoxygenated to study the effects of myocardial hypoxia. A subset of hearts was perfused with 1 μm tolbutamide (TOLB) to identify the level of AP duration (APD) shortening attributed to KATP channel activation. During sinus rhythm, APD was shorter in LWHs compared to LANG hearts. APD in both LWHs and LANG hearts dropped steadily during deoxygenation. With TOLB, APDs in LWHs were longer at all workloads and APD reductions during deoxygenation were blunted in both LWHs and LANG hearts. At 50% perfusate oxygenation, APD and LVDP were significantly higher in LWHs perfused with TOLB (199 ± 16 ms; 92 ± 5.3 mmHg) than in LWHs without TOLB (109 ± 14 ms, P = 0.005; 65 ± 6.5 mmHg, P = 0.01). Our results indicate that KATP channels are activated to a greater extent in perfused hearts when the LV performs pressure–volume work. The results of the present study demonstrate the critical role of KATP channels in modulating myocardial function over a wide range of physiological conditions.

Key points
How parallel are the primate visual pathways? In the present study, we demonstrate that parallel visual pathways in the dorsal lateral geniculate nucleus (LGN) show distinct patterns of interaction with rhythmic activity in the primary visual cortex (V1).
In the V1 of anaesthetized marmosets, the EEG frequency spectrum undergoes transient changes that are characterized by fluctuations in delta‐band EEG power.
We show that, on multisecond timescales, spiking activity in an evolutionary primitive (koniocellular) LGN pathway is specifically linked to these slow EEG spectrum changes. By contrast, on subsecond (delta frequency) timescales, cortical oscillations can entrain spiking activity throughout the entire LGN.
Our results are consistent with the hypothesis that, in waking animals, the koniocellular pathway selectively participates in brain circuits controlling vigilance and attention.
Abstract
The major afferent cortical pathway in the visual system passes through the dorsal lateral geniculate nucleus (LGN), where nerve signals originating in the eye can first interact with brain circuits regulating visual processing, vigilance and attention. In the present study, we investigated how ongoing and visually driven activity in magnocellular (M), parvocellular (P) and koniocellular (K) layers of the LGN are related to cortical state. We recorded extracellular spiking activity in the LGN simultaneously with local field potentials (LFP) in primary visual cortex, in sufentanil‐anaesthetized marmoset monkeys. We found that asynchronous cortical states (marked by low power in delta‐band LFPs) are linked to high spike rates in K cells (but not P cells or M cells), on multisecond timescales. Cortical asynchrony precedes the increases in K cell spike rates by 1–3 s, implying causality. At subsecond timescales, the spiking activity in many cells of all (M, P and K) classes is phase‐locked to delta waves in the cortical LFP, and more cells are phase‐locked during synchronous cortical states than during asynchronous cortical states. The switch from low‐to‐high spike rates in K cells does not degrade their visual signalling capacity. By contrast, during asynchronous cortical states, the fidelity of visual signals transmitted by K cells is improved, probably because K cell responses become less rectified. Overall, the data show that slow fluctuations in cortical state are selectively linked to K pathway spiking activity, whereas delta‐frequency cortical oscillations entrain spiking activity throughout the entire LGN, in anaesthetized marmosets.

Key points
Pharmacological and molecular inhibition of transient receptor potential melastatin 7 (TRPM7) reduces store‐operated calcium entry (SOCE).
Overexpression of TRPM7 in TRPM7−/− cells restores SOCE.
TRPM7 is not a store‐operated calcium channel.
TRPM7 kinase rather than channel modulates SOCE.
TRPM7 channel activity contributes to the maintenance of store Ca2+ levels at rest.
Abstract
The transient receptor potential melastatin 7 (TRPM7) is a protein that combines an ion channel with an intrinsic kinase domain, enabling it to modulate cellular functions either by conducting ions through the pore or by phosphorylating downstream proteins via its kinase domain. In the present study, we report store‐operated calcium entry (SOCE) as a novel target of TRPM7 kinase activity. TRPM7‐deficient chicken DT40 B lymphocytes exhibit a strongly impaired SOCE compared to wild‐type cells as a result of reduced calcium release activated calcium currents, and independently of potassium channel regulation, membrane potential changes or changes in cell‐cycle distribution. Pharmacological blockade of TRPM7 with NS8593 or waixenicin A in wild‐type B lymphocytes results in a significant decrease in SOCE, confirming that TRPM7 activity is acutely linked to SOCE, without TRPM7 representing a store‐operated channel itself. Using kinase‐deficient mutants, we find that TRPM7 regulates SOCE through its kinase domain. Furthermore, Ca2+ influx through TRPM7 is essential for the maintenance of endoplasmic reticulum Ca2+ concentration in resting cells, and for the refilling of Ca2+ stores after a Ca2+ signalling event. We conclude that the channel kinase TRPM7 and SOCE are synergistic mechanisms regulating intracellular Ca2+ homeostasis.

Key points
Chronic perinatal nicotine exposure causes abnormal auditory brainstem responses and auditory processing deficits in children and animal models.
The effect of perinatal nicotine exposure on synaptic maturation in the auditory brainstem was investigated in granule cells in the ventral nucleus of the lateral lemniscus, which receive a single calyx‐like input from the cochlear nucleus.
Perinatal nicotine exposure caused a massive reduction in the amplitude of the excitatory input current.
This caused a profound decrease in the number and temporal precision of spikes in these neurons.
Perinatal nicotine exposure delayed the developmental downregulation of functional nicotinic acetylcholine receptors on these neurons.
Abstract
Maternal smoking causes chronic nicotine exposure during early development and results in auditory processing deficits including delayed speech development and learning difficulties. Using a mouse model of chronic, perinatal nicotine exposure we explored to what extent synaptic inputs to granule cells in the ventral nucleus of the lateral lemniscus are affected by developmental nicotine treatment. These neurons receive one large calyx‐like input from octopus cells in the cochlear nucleus and play a role in sound pattern analysis, including speech sounds. In addition, they exhibit high levels of α7 nicotinic acetylcholine receptors, especially during early development. Our whole‐cell patch‐clamp experiments show that perinatal nicotine exposure causes a profound reduction in synaptic input amplitude. In contrast, the number of inputs innervating each neuron and synaptic release properties of this calyx‐like synapse remained unaltered. Spike number and spiking precision in response to synaptic stimulation were greatly diminished, especially for later stimuli during a stimulus train. Moreover, chronic nicotine exposure delayed the developmental downregulation of functional nicotinic acetylcholine receptors on these neurons, indicating a direct action of nicotine in this brain area. This presumably direct effect of perinatal nicotine exposure on synaptic maturation in the auditory brainstem might be one of the underlying causes for auditory processing difficulties in children of heavy smoking mothers.

Key points
Severe aortic valve diseases are common cardiac abnormalities that are associated with poor long‐term survival.
Before any reduction in left ventricular (LV) function, the left ventricle undergoes structural remodelling under the influence of changing haemodynamic conditions.
In this study, we combined temporal changes in LV structure (volume) with alterations in LV functional characteristics (strain, ԑ) into a ԑ–volume loop, in order to provide novel insight into the haemodynamic cardiac consequences of aortic valve diseases in those with preserved LV ejection fraction.
We showed that our novel ԑ–volume loop and the specific loop characteristics provide additional insight into the functional and mechanical haemodynamic consequences of severe aortic valve diseases (with preserved LV ejection fraction).
Finally, we showed that the ԑ–volume loop characteristics provide discriminative capacity compared with conventional measures of LV function.
Abstract
The purpose of this study was to examine left ventricular (LV) strain (ԑ)–volume loops to provide novel insight into the haemodynamic cardiac consequences of aortic valve stenosis (AS) and aortic valve regurgitation (AR). Twenty‐seven participants were retrospectively recruited: AR (n = 7), AS (n = 10) and control subjects (n = 10). Standard transthoracic echocardiography was used to obtain apical four‐chamber images to construct ԑ–volume relationships, which were assessed using the following parameters: early systolic ԑ (ԑ_ES); slope of ԑ–volume relationship during systole (Sslope); end‐systolic peak ԑ (peak ԑ); and diastolic uncoupling (systolic ԑ–diastolic ԑ at same volume) during early diastole (UNCOUP_ED) and late diastole (UNCOUP_LD). Receiver operating characteristic curves were used to determine the ability to detect impaired LV function. Although LV ejection fraction was comparable between groups, longitudinal peak ԑ was reduced compared with control subjects. In contrast, ԑ_ES and Sslope were lower in both pathologies compared with control subejcts (P 0.05) to distinguish AS vs control, AR vs control and AS vs AR groups, respectively. Temporal changes in ԑ–volume characteristics provide novel insight into the haemodynamic cardiac impact of AS and AR. Contrary to traditional measures (i.e. ejection fraction, peak ԑ), these novel measures successfully distinguish between the haemodynamic cardiac impact of AS and AR.

Key points
Mouse chromaffin cells (MCCs) generate spontaneous burst‐firing that causes large increases of Ca2+‐dependent catecholamine release, and is thus a key mechanism for regulating the functions of MCCs.
With the aim to uncover a physiological role for burst‐firing we investigated the effects of acidosis on MCC activity.
Lowering the extracellular pH (pHo) from 7.4 to 6.6 induces cell depolarizations of 10–15 mV that generate bursts of ∼330 ms at 1–2 Hz and a 7.4‐fold increase of cumulative catecholamine‐release.
Burst‐firing originates from the inhibition of the pH‐sensitive TASK‐1‐channels and a 60% reduction of BK‐channel conductance at pHo 6.6.
Blockers of the two channels (A1899 and paxilline) mimic the effects of pHo 6.6, and this is reverted by the Cav1 channel blocker nifedipine.
MCCs act as pH‐sensors. At low pHo, they depolarize, undergo burst‐firing and increase catecholamine‐secretion, generating an effective physiological response that may compensate for the acute acidosis and hyperkalaemia generated during heavy exercise and muscle fatigue.
Abstract
Mouse chromaffin cells (MCCs) generate action potential (AP) firing that regulates the Ca2+‐dependent release of catecholamines (CAs). Recent findings indicate that MCCs possess a variety of spontaneous firing modes that span from the common ‘tonic‐irregular’ to the less frequent ‘burst’ firing. This latter is evident in a small fraction of MCCs but occurs regularly when Nav1.3/1.7 channels are made less available or when the Slo1β2‐subunit responsible for BK channel inactivation is deleted. Burst firing causes large increases of Ca2+‐entry and potentiates CA release by ∼3.5‐fold and thus may be a key mechanism for regulating MCC function. With the aim to uncover a physiological role for burst‐firing we investigated the effects of acidosis on MCC activity. Lowering the extracellular pH (pHo) from 7.4 to 7.0 and 6.6 induces cell depolarizations of 10–15 mV that generate repeated bursts. Bursts at pHo 6.6 lasted ∼330 ms, occurred at 1–2 Hz and caused an ∼7‐fold increase of CA cumulative release. Burst firing originates from the inhibition of the pH‐sensitive TASK‐1/TASK‐3 channels and from a 40% BK channel conductance reduction at pHo 7.0. The same pHo had little or no effect on Nav, Cav, Kv and SK channels that support AP firing in MCCs. Burst firing of pHo 6.6 could be mimicked by mixtures of the TASK‐1 blocker A1899 (300 nm) and BK blocker paxilline (300 nm) and could be prevented by blocking L‐type channels by adding 3 μm nifedipine. Mixtures of the two blockers raised cumulative CA‐secretion even more than low pHo (∼12‐fold), showing that the action of protons on vesicle release is mainly a result of the ionic conductance changes that increase Ca2+‐entry during bursts. Our data provide direct evidence suggesting that MCCs respond to low pHo with sustained depolarization, burst firing and enhanced CA‐secretion, thus mimicking the physiological response of CCs to acute acidosis and hyperkalaemia generated during heavy exercise and muscle fatigue.

Key points
Voltage‐gated sodium channels play a fundamental role in determining neuronal excitability.
Specifically, voltage‐gated sodium channel subtype NaV1.7 is required for sensing acute and inflammatory somatic pain in mice and humans but its significance in pain originating from the viscera is unknown.
Using comparative behavioural models evoking somatic and visceral pain pathways, we identify the requirement for NaV1.7 in regulating somatic (noxious heat pain threshold) but not in visceral pain signalling.
These results enable us to better understand the mechanisms underlying the transduction of noxious stimuli from the viscera, suggest that the investigation of pain pathways should be undertaken in a modality‐specific manner and help to direct drug discovery efforts towards novel visceral analgesics.
Abstract
Voltage‐gated sodium channel NaV1.7 is required for acute and inflammatory pain in mice and humans but its significance for visceral pain is unknown. Here we examine the role of NaV1.7 in visceral pain processing and the development of referred hyperalgesia using a conditional nociceptor‐specific NaV1.7 knockout mouse (NaV1.7Nav1.8) and selective small‐molecule NaV1.7 antagonist PF‐5198007. NaV1.7Nav1.8 mice showed normal nociceptive behaviours in response to intracolonic application of either capsaicin or mustard oil, stimuli known to evoke sustained nociceptor activity and sensitization following tissue damage, respectively. Normal responses following induction of cystitis by cyclophosphamide were also observed in both NaV1.7Nav1.8 and littermate controls. Loss, or blockade, of NaV1.7 did not affect afferent responses to noxious mechanical and chemical stimuli in nerve–gut preparations in mouse, or following antagonism of NaV1.7 in resected human appendix stimulated by noxious distending pressures. However, expression analysis of voltage‐gated sodium channel α subunits revealed NaV1.7 mRNA transcripts in nearly all retrogradely labelled colonic neurons, suggesting redundancy in function. By contrast, using comparative somatic behavioural models we identify that genetic deletion of NaV1.7 (in NaV1.8‐expressing neurons) regulates noxious heat pain threshold and that this can be recapitulated by the selective NaV1.7 antagonist PF‐5198007. Our data demonstrate that NaV1.7 (in NaV1.8‐expressing neurons) contributes to defined pain pathways in a modality‐dependent manner, modulating somatic noxious heat pain, but is not required for visceral pain processing, and advocate that pharmacological block of NaV1.7 alone in the viscera may be insufficient in targeting chronic visceral pain.

Key points
Pre‐ischaemic administration of aminooxiacetate (AOA), an inhibitor of the malate‐aspartate shuttle (MAS), provides cardioprotection against ischaemia–reperfusion injury. The underlying mechanism remains unknown.
We examined whether transient inhibition of the MAS during ischaemia and early reperfusion by AOA treatment could prevent mitochondrial damage at later reperfusion.
The AOA treatment preserved mitochondrial respiratory capacity with reduced mitochondrial oxidative stress during late reperfusion to the same extent as ischaemic preconditioning (IPC).
However, AOA treatment, but not IPC, reduced the myocardial interstitial concentration of tricarboxylic acid cycle intermediates at the onset of reperfusion.
The results obtained in the present study demonstrate that metabolic regulation by inhibition of the MAS at the onset of reperfusion may be beneficial for the preservation of mitochondrial function during late reperfusion in an IR‐injured heart.
Abstract
Mitochondrial dysfunction plays a central role in ischaemia–reperfusion (IR) injury. Pre‐ischaemic administration of aminooxyacetate (AOA), an inhibitor of the malate‐aspartate shuttle (MAS), provides cardioprotection against IR injury, although the underlying mechanism remains unknown. We hypothesized that a transient inhibition of the MAS during ischaemia and early reperfusion could preserve mitochondrial function at later phase of reperfusion in the IR‐injured heart to the same extent as ischaemic preconditioning (IPC), which is a well‐validated cardioprotective strategy against IR injury. In the present study, we show that pre‐ischaemic administration of AOA preserved mitochondrial complex I‐linked state 3 respiration and fatty acid oxidation during late reperfusion in IR‐injured isolated rat hearts. AOA treatment also attenuated the excessive emission of mitochondrial reactive oxygen species during state 3 with complex I‐linked substrates during late reperfusion, which was consistent with reduced oxidative damage in the IR‐injured heart. As a result, AOA treatment reduced infarct size after reperfusion. These protective effects of MAS inhibition on the mitochondria were similar to those of IPC. Intriguingly, the protection of mitochondrial function by AOA treatment appears to be different from that of IPC because AOA treatment, but not IPC, downregulated myocardial tricarboxilic acid (TCA)‐cycle intermediates at the onset of reperfusion. MAS inhibition thus preserved mitochondrial respiratory capacity and decreased mitochondrial oxidative stress during late reperfusion in the IR‐injured heart, at least in part, via metabolic regulation of TCA cycle intermediates in the mitochondria at the onset of reperfusion.

Key points
Cardiac sympathetic afferents are considered to be essential pathways for transmission of cardiac nociception to the central nervous system during myocardial ischaemia. However, a potential contribution of the CSAR control of cardiac dysfunction in both normal and chronic heart failure (CHF) states remains unknown.
We found that activation of the CSAR evokes little increase in cardiac contractility with an exaggerated peripheral vasoconstriction in the CHF state.
CSAR inhibition by epicardial lidocaine decreased cardiac contractility to a greater extent in CHF rats than sham rats. Furthermore, we also found that epicardial lidocaine paradoxically decreased left ventricular end‐diastolic pressure (LVEDP) and left ventricular end‐diastolic volume (preload) in CHF rats, which was not observed in sham rats.
Chronic ablation of the CSAR by epicardial application of the afferent neurotoxin, RTX, selectively lowered diastolic blood pressure CHF rats.
The observation suggests that CSAR has a differential effect on cardiac function in normal and CHF states. CSAR activation in normal state causes significant increase in cardiac contractility and cardiac output.
Abstract
The enhanced ‘cardiac sympathetic afferent reflex’ (CSAR) critically contributes to the exaggerated global sympathetic tone in chronic heart failure (CHF). However, a potential contribution of the cardio‐cardiac reflex control of cardiac function in both normal and CHF states remains unknown. In this study, we evaluated the effects of direct activation or inhibition of the CSAR on cardiac function by pressure–volume (P–V) loop analysis in ∼12‐week sham‐operated and myocardial infarcted (MI) rats. In sham rats, acute CSAR activation by epicardial application of bradykinin (BK) increased heart rate (HR), left ventricular systolic pressure (LVSP), the maximum first derivative of left ventricular pressure (dp/dtmax), and the slope of the end‐systolic P–V relationship (ESPVR), suggesting that acute CSAR activation in the normal state enhances myocardial contractility. CSAR activation also decreased left ventricular (LV) systolic and diastolic volumes with little effect on LV end‐diastolic pressure (LVEDP) or the end‐diastolic P–V relationship (EDPVR) in sham rats. Compared to sham, CHF rats exhibit a reduced increase in the slope of the ESPVR and dp/dtmax in response to BK, indicating a poor contractile response to CSAR activation. Interestingly, BK application in CHF rats increased cardiac systolic and diastolic volumes and further increased the elevated LVEDP, neither of which was seen in sham rats. Following CSAR inhibition by epicardial lidocaine, blood pressure, HR, LVSP, dp/dt, LVEDP and ESPVR decreased in CHF rats whereas lidocaine had little effect in sham rats, indicating that the CSAR is tonically active in CHF and contributes to cardiac dysfunction. Furthermore, we found that epicardial lidocaine paradoxically decreased LV end‐diastolic volume (preload) in CHF rats, which was not observed in sham rats. The decreased preload by lidocaine in CHF rats may be due to a reduction in peripheral vascular resistance since epicardial lidocaine significantly lowered peripheral (renal) sympathetic nerve activity in CHF rats but not in sham rats. Furthermore, chronic ablation of CSAR by epicardial application of a selective afferent neurotoxin, resiniferatoxin, selectively lowered diastolic blood pressure both at daytime and night‐time with less effect on systolic blood pressure in CHF rats. Our data suggest that there is an imbalance between cardiac and peripheral responses to CSAR in CHF animals compared to sham‐operated controls.

Key points
Relaxin‐3 is a stress‐responsive neuropeptide that acts at its cognate receptor, RXFP3, to alter behaviours including feeding. In this study, we have demonstrated a direct, RXFP3‐dependent, inhibitory action of relaxin‐3 on oxytocin and vasopressin paraventricular nucleus (PVN) neuron electrical activity, a putative cellular mechanism of orexigenic actions of relaxin‐3.
We observed a Gαi/o‐protein‐dependent inhibitory influence of selective RXFP3 activation on PVN neuronal activity in vitro and demonstrated a direct action of RXFP3 activation on oxytocin and vasopressin PVN neurons, confirmed by their abundant expression of RXFP3
mRNA. Moreover, we demonstrated that RXFP3 activation induces a cadmium‐sensitive outward current, which indicates the involvement of a characteristic magnocellular neuron outward potassium current.
Furthermore, we identified an abundance of relaxin‐3‐immunoreactive axons/fibres originating from the nucleus incertus in close proximity to the PVN, but associated with sparse relaxin‐3‐containing fibres/terminals within the PVN.
Abstract
The paraventricular nucleus of the hypothalamus (PVN) plays an essential role in the control of food intake and energy expenditure by integrating multiple neural and humoral inputs. Recent studies have demonstrated that intracerebroventricular and intra‐PVN injections of the neuropeptide relaxin‐3 or selective relaxin‐3 receptor (RXFP3) agonists produce robust feeding in satiated rats, but the cellular and molecular mechanisms of action associated with these orexigenic effects have not been identified. In the present studies, using rat brain slices, we demonstrated that relaxin‐3, acting through its cognate G‐protein‐coupled receptor, RXFP3, hyperpolarized a majority of putative magnocellular PVN neurons (88%, 22/25), including cells producing the anorexigenic neuropeptides, oxytocin and vasopressin. Importantly, the action of relaxin‐3 persisted in the presence of tetrodotoxin and glutamate/GABA receptor antagonists, indicating its direct action on PVN neurons. Similar inhibitory effects on PVN oxytocin and vasopressin neurons were produced by the RXFP3 agonist, RXFP3‐A2 (82%, 80/98 cells). In situ hybridization histochemistry revealed a strong colocalization of RXFP3 mRNA with oxytocin and vasopressin immunoreactivity in rat PVN neurons. A smaller percentage of putative parvocellular PVN neurons was sensitive to RXFP3‐A2 (40%, 16/40 cells). These data, along with a demonstration of abundant peri‐PVN and sparse intra‐PVN relaxin‐3‐immunoreactive nerve fibres, originating from the nucleus incertus, the major source of relaxin‐3 neurons, identify a strong inhibitory influence of relaxin‐3–RXFP3 signalling on the electrical activity of PVN oxytocin and vasopressin neurons, consistent with the orexigenic effect of RXFP3 activation observed in vivo.

Key points
The corticospinal tract contributes to the control of finger muscles during precision and power grip. We explored the neural mechanisms contributing to changes in corticospinal excitability during these gripping configurations.
Motor evoked potentials (MEPs) elicited by cortical, but not by subcortical, stimulation were more suppressed during power grip compared with precision grip and index finger abduction.
Intracortical inhibition was more reduced during power grip compared with the other tasks. An acoustic startle cue, a stimulus that engages the reticular system, suppressed MEP size during power grip to a lesser extent than during the other tasks at a cortical level and this positively correlated with changes in intracortical inhibition.
Our findings suggest that changes in corticospinal excitability during gross more than fine finger manipulations are largely cortical in origin and that the reticular system contributed, at least in part, to these effects.
Abstract
It is well accepted that the corticospinal tract contributes to the control of finger muscles during precision and power grip in humans but the neural mechanisms involved remain poorly understood. Here, we examined motor evoked potentials elicited by cortical and subcortical stimulation of corticospinal axons (MEPs and CMEPs, respectively) and the activity in intracortical circuits (suppression of voluntary electromyography) and spinal motoneurons (F‐waves) in an intrinsic hand muscle during index finger abduction, precision grip and power grip. We found that the size of MEPs, but not CMEPs, was more suppressed during power grip compared with precision grip and index finger abduction, suggesting a cortical origin for these effects. Notably, intracortical inhibition was more reduced during power grip compared with the other tasks. To further examine the origin of changes in intracortical inhibition we assessed the contribution of the reticular system, which projects to cortical neurons, and projects to spinal motoneurons controlling hand muscles. An acoustic startle cue, which engages the reticular system, suppressed MEP size during power grip to a lesser extent than during the other tasks and this positively correlated with changes in intracortical inhibition. A startle cue decreased intracortical inhibition, but not CMEPs, during power grip. F‐waves remained unchanged across conditions. Our novel findings show that changes in corticospinal excitability present during power grip compared with fine finger manipulations are largely cortical in origin and suggest that the reticular system contributed, at least in part, to these effects.

Key points
The role of enteric glial cell activity in the acute regulation of epithelial barrier and secretomotor functions of the intestines under physiological conditions is not clear.
We used transgenic mice to modify glial activity and found that enteric glia significantly contribute to the neurogenic ion transport while glial activity does not appear to play a major role in the acute regulation of barrier function.
The selective activation of glial activity evoked electrogenic ion transport primarily through neural pathways and was sufficient to drive electrogenic ion transport to an extent equal to the direct activation of neurogenic ion transport.
These findings provide novel insight into the cellular mechanisms that control fluid transport homeostasis in the intestine and might provide novel therapeutic avenues for functional diarrheal diseases.
Abstract
Enteric glial cells are often implicated in the regulation of epithelial barrier and secretomotor functions of the intestines. But whether glial cell activity regulates these functions acutely under physiological conditions is not clear. We addressed this issue by using transgenic animal models to modify the activity of enteric glia, either reducing glial expression of connexin 43 in Sox10::CreERT2+/−/Cx43f/f mice or activating glial calcium responses in GFAP::hM3Dq mice, and tested the effects on colonic barrier function and electrogenic ion transport in Ussing chambers. We assessed neuronal‐dependent and ‐independent contributions by activating or inhibiting neurogenic activity with veratridine and tetrodotoxin, respectively. Our results show that the reduction of glial Cx43 expression in Sox10::CreERT2+/−/Cx43f/f mice significantly reduced neurogenic ion transport. The selective glial activation in tissues from GFAP::hM3Dq mice evoked electrogenic ion transport to an extent equal to the direct activation of neurogenic ion transport with veratridine and glial driven responses consisted of both tetrodotoxin‐sensitive and ‐insensitive components. The selective glial stimulation did not affect transmural ion conductance or cell‐impermeant dye flux but the baseline ion conductance was more variable in Sox10::CreERT2+/−/Cx43f/f tissues. Together, our findings show that glial activity contributes to the regulation of electrogenic ion transport in the intestine through effects on neurons and possibly direct effects on epithelial cells. However, glial activity does not appear to play a major role in the acute regulation of barrier function. These findings provide novel insight into the cellular mechanisms that control fluid transport homeostasis in the intestine.

Key points
The transient receptor potential vanilloid type 1 (TRPV1) receptor is a polymodal molecular integrator in the pain pathway expressed in Aδ‐ and C‐fibre nociceptors and is responsible for the thermal hyperalgesia associated with inflammatory pain.
Noradrenaline strongly inhibited the activity of TRPV1 channels in dorsal root ganglia neurons. The effect of noradrenaline was reproduced by clonidine and antagonized by yohimbine, consistent with contribution of α2 adrenergic receptors.
The inhibitory effect of noradrenaline on TRPV1 channels was dependent on calcium influx and linked to calcium/calmodulin‐dependent protein kinase II.
In spinal cord slices, clonidine reduced the frequency of capsaicin‐induced miniature EPSCs in the presence of tetrodotoxin and ω‐conotoxin‐MVIIC, consistent with inhibition of presynaptic TRPV1 channels by α2 adrenergic receptors.
We suggest that modulation of presynaptic TRPV1 channels in nociceptive neurons by descending noradrenergic inputs may constitute a mechanism for noradrenaline to modulate incoming noxious stimuli in the dorsal horn of the spinal cord.
Abstract
The transient receptor potential vanilloid type 1 (TRPV1) receptor is a well‐known contributor to nociceptor excitability. To address whether noradrenaline can down‐regulate TRPV1 channel activity in nociceptors and reduce their synaptic transmission, the effects of noradrenaline and clonidine were tested on the capsaicin‐activated current recorded from acutely dissociated small diameter (<27 μm) dorsal root ganglia (DRG) neurons and on miniature (m)EPSCs recorded from large lamina I neurons in horizontal spinal cord slices. Noradrenaline or clonidine inhibited the capsaicin‐activated current by ∼60%, and the effect was reversed by yohimbine, confirming that it was mediated by activation of α2 adrenergic receptors. Similarly, clonidine reduced the frequency of capsaicin‐induced mEPSCs by ∼60%. Inhibition of capsaicin‐activated current by noradrenaline was mediated by GTP binding proteins, and was highly dependent on calcium influx. The inhibitory effect of noradrenaline on the capsaicin‐activated current was not affected either by blocking the activity of protein kinase A with H89, or by blocking the activity of protein kinase C with bisindolylmaleimide II. In contrast, when the calcium/calmodulin‐dependent protein kinase II (CaMKII) was blocked with KN‐93, the inhibitory effect of noradrenaline on the capsaicin‐activated current was greatly reduced, suggesting that activation of adrenergic receptors in DRG neurons is preferentially linked to CaMKII activity. We suggest that modulation of TRPV1 channels by noradrenaline in nociceptive neurons is a mechanism whereby noradrenaline may suppress incoming noxious stimuli at the primary synaptic afferents in the dorsal horn of the spinal cord.

Key points
The vestibular influence on human walking is phase‐dependent and modulated across both limbs with changes in locomotor velocity and cadence.
Using a split‐belt treadmill, we show that vestibular influence on locomotor activity is modulated independently in each limb.
The independent vestibular modulation of muscle activity from each limb occurs rapidly at the onset of split‐belt walking, over a shorter time course relative to the characteristic split‐belt error‐correction mechanisms (i.e. muscle activity and kinematics) associated with locomotor adaptation.
Together, the present results indicate that the nervous system rapidly modulates the vestibular influence of each limb separately through processes involving ongoing sensory feedback loops.
These findings help us understand how vestibular information is used to accommodate the variable and commonplace demands of locomotion, such as turning or navigating irregular terrain.
Abstract
During walking, the vestibular influence on locomotor activity is phase‐dependent and modulated in both limbs with changes in velocity. It is unclear, however, whether this bilateral modulation is due to a coordinated mechanism between both limbs or instead through limb‐specific processes that remain masked by the symmetric nature of locomotion. Here, human subjects walked on a split‐belt treadmill with one belt moving at 0.4 m s−1 and the other moving at 0.8 m s−1 while exposed to an electrical vestibular stimulus. Muscle activity was recorded bilaterally around the ankles of each limb and used to compare vestibulo‐muscular coupling between velocity‐matched and unmatched tied‐belt walking. In general, response magnitudes decreased by ∼20–50% and occurred ∼13–20% earlier in the stride cycle at the higher belt velocity. This velocity‐dependent modulation of vestibular‐evoked muscle activity was retained during split‐belt walking and was similar, within each limb, to velocity‐matched tied‐belt walking. These results demonstrate that the vestibular influence on ankle muscles during locomotion can be adapted independently to each limb. Furthermore, modulation of vestibular‐evoked muscle responses occurred rapidly (∼13–34 strides) after onset of split‐belt walking. This rapid adaptation contrasted with the prolonged adaptation in step length symmetry (∼128 strides) as well as EMG magnitude and timing (∼40–100 and ∼20–70 strides, respectively). These results suggest that vestibular influence on ankle muscle control is adjusted rapidly in sensorimotor control loops as opposed to longer‐term error correction mechanisms commonly associated with split‐belt adaptation. Rapid limb‐specific sensorimotor feedback adaptation may be advantageous for asymmetric overground locomotion, such as navigating irregular terrain or turning.

Key points
In order to understand how the brain's coding strategies are adapted to the statistics of the sensory stimuli experienced during everyday life, the use of animal models is essential.
Mice and non‐human primates have become common models for furthering our knowledge of the neuronal coding of natural stimuli, but differences in their natural environments and behavioural repertoire may impact optimal coding strategies.
Here we investigated the structure and statistics of the vestibular input experienced by mice versus non‐human primates during natural behaviours, and found important differences.
Our data establish that the structure and statistics of natural signals in non‐human primates more closely resemble those observed previously in humans, suggesting similar coding strategies for incoming vestibular input.
These results help us understand how the effects of active sensing and biomechanics will differentially shape the statistics of vestibular stimuli across species, and have important implications for sensory coding in other systems.
Abstract
It is widely believed that sensory systems are adapted to the statistical structure of natural stimuli, thereby optimizing coding. Recent evidence suggests that this is also the case for the vestibular system, which senses self‐motion and in turn contributes to essential brain functions ranging from the most automatic reflexes to spatial perception and motor coordination. However, little is known about the statistics of self‐motion stimuli actually experienced by freely moving animals in their natural environments. Accordingly, here we examined the natural self‐motion signals experienced by mice and monkeys: two species commonly used to study vestibular neural coding. First, we found that probability distributions for all six dimensions of motion (three rotations, three translations) in both species deviated from normality due to long tails. Interestingly, the power spectra of natural rotational stimuli displayed similar structure for both species and were not well fitted by power laws. This result contrasts with reports that the natural spectra of other sensory modalities (i.e. vision, auditory and tactile) instead show a power‐law relationship with frequency, which indicates scale invariance. Analysis of natural translational stimuli revealed important species differences as power spectra deviated from scale invariance for monkeys but not for mice. By comparing our results to previously published data for humans, we found the statistical structure of natural self‐motion stimuli in monkeys and humans more closely resemble one another. Our results thus predict that, overall, neural coding strategies used by vestibular pathways to encode natural self‐motion stimuli are fundamentally different in rodents and primates.

Key points
Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle.
The magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiac myocytes are largely unknown.
Rapid stimulation frequency‐dependent increases but relatively slow decreases in free mitochondrial calcium concentration were observed in rat cardiac myocytes. This asymmetry caused a rise in the mitochondrial calcium concentration with stimulation frequency.
These results provide insight into the mechanisms of mitochondrial calcium uptake and release that are important in healthy and diseased myocardium.
Abstract
Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle. Little is known about the magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiomyocytes. Using adenoviral infection, a ratiometric mitochondrially targeted Förster resonance energy transfer (FRET)‐based calcium indicator (4mtD3cpv, MitoCam) was expressed in cultured adult rat cardiomyocytes and the free mitochondrial calcium concentration ([Ca2+]m) was measured at different stimulation frequencies (0.1–4 Hz) and external calcium concentrations (1.8–3.6 mm) at 37°C. Cytosolic calcium concentrations were assessed under the same experimental conditions in separate experiments using Fura‐4AM. The increases in [Ca2+]m during electrical stimulation at 0.1 Hz were rapid (rise time = 49 ± 2 ms), while the decreases in [Ca2+]m occurred more slowly (decay half time = 1.17 ± 0.07 s). Model calculations confirmed that this asymmetry caused the rise in [Ca2+]m during diastole observed at elevated stimulation frequencies. Inhibition of the mitochondrial sodium–calcium exchanger (mNCE) resulted in a rise in [Ca2+]m at baseline and, paradoxically, in an acceleration of Ca2+ release. In conclusion: rapid increases in [Ca2+]m allow for fast adjustment of mitochondrial ATP production to increases in myocardial demand on a beat‐to‐beat basis and mitochondrial calcium release depends on mNCE activity and mitochondrial calcium buffering.

Key points
The principles underlying the evolutionary selection of ion channels for expression in sensory neurons are unclear.
Photoreceptor depolarization in the diurnal Drosophila melanogaster is predominantly provided by light‐activated transient receptor potential (TRP) channels, whereas repolarization is mediated by sustained voltage‐gated K+ channels of the Shab family.
In the present study, we show that phototransduction in the nocturnal cockroach Periplaneta americana is predominantly mediated by TRP‐like channels, whereas membrane repolarization is based on EAG channels.
Although bright light stimulates Shab channels in Drosophila, further restricting depolarization and improving membrane bandwidth, it strongly suppresses EAG conductance in Periplaneta. This light‐dependent inhibition (LDI) is caused by calcium and is abolished by chelating intracellular calcium or suppressing eag gene expression.
LDI increases membrane resistance, augments gain and reduces the signalling bandwidth. This makes EAG unsuitable for light response conditioning during the day and might have resulted in the evolutionary replacement of EAG by other delayed rectifiers in diurnal insects.
Abstract
The principles underlying evolutionary selection of ion channels for expression in sensory neurons are unclear. Among species possessing microvillar photoreceptors, the major ionic conductances have only been identified in Drosophila melanogaster. In Drosophila, depolarization is provided by light‐activated transient receptor potential (TRP) channels with a minor contribution from TRP‐like (TRPL) channels, whereas repolarization is mediated by sustained voltage‐gated K+ (Kv) channels of the Shab family. Bright light stimulates Shab channels, further restricting depolarization and improving membrane bandwidth. In the present study, data obtained using a combination of electrophysiological, pharmacological and molecular knockdown techniques strongly suggest that in photoreceptors of the nocturnal cockroach Periplaneta americana the major excitatory channel is TRPL, whereas the predominant delayed rectifier is EAG, a ubiquitous but enigmatic Kv channel. By contrast to the diurnal Drosophila, bright light strongly suppresses EAG conductance in Periplaneta. This light‐dependent inhibition (LDI) is caused by calcium entering the cytosol and is amplified following inhibition of calcium extrusion, and it can also be abolished by chelating intracellular calcium or suppressing eag gene expression by RNA interference. LDI increases membrane resistance, augments gain and reduces the signalling bandwidth, impairing information transfer. LDI is also observed in the nocturnal cricket Gryllus integer, whereas, in the diurnal water strider Gerris lacustris, the delayed rectifier is up‐regulated by light. Although LDI is not expected to reduce delayed rectifier current in the normal illumination environment of nocturnal cockroaches and crickets, it makes EAG unsuitable for light response conditioning during the day, and might have resulted in the evolutionary replacement of EAG by other delayed rectifiers in diurnal insects.

Key points
Late gestation during pregnancy has been associated with a relatively high prevalence of obstructive sleep apnoea (OSA).
Intermittent hypoxia, a hallmark of OSA, could impose significant long‐term effects on somatic growth, energy homeostasis and metabolic function in offspring.
Here we show that late gestation intermittent hypoxia induces metabolic dysfunction as reflected by increased body weight and adiposity index in adult male offspring that is paralleled by epigenomic alterations and inflammation in visceral white adipose tissue.
Fetal perturbations by OSA during pregnancy impose long‐term detrimental effects manifesting as metabolic dysfunction in adult male offspring.
Abstract
Pregnancy, particularly late gestation (LG), has been associated with a relatively high prevalence of obstructive sleep apnoea (OSA). Intermittent hypoxia (IH), a hallmark of OSA, could impose significant long‐term effects on somatic growth, energy homeostasis, and metabolic function in offspring. We hypothesized that IH during late pregnancy (LG‐IH) may increase the propensity for metabolic dysregulation and obesity in adult offspring via epigenetic modifications. Time‐pregnant female C57BL/6 mice were exposed to LG‐IH or room air (LG‐RA) during days 13–18 of gestation. At 24 weeks, blood samples were collected from offspring mice for lipid profiles and insulin resistance, indirect calorimetry was performed and visceral white adipose tissues (VWAT) were assessed for inflammatory cells as well as for differentially methylated gene regions (DMRs) using a methylated DNA immunoprecipitation on chip (MeDIP‐chip). Body weight, food intake, adiposity index, fasting insulin, triglycerides and cholesterol levels were all significantly higher in LG‐IH male but not female offspring. LG‐IH also altered metabolic expenditure and locomotor activities in male offspring, and increased number of pro‐inflammatory macrophages emerged in VWAT along with 1520 DMRs (P

Key points
When a skeletal muscle is stretched while it contracts, the muscle produces a relatively higher force than the force from an isometric contraction at the same length: a phenomenon referred to as residual force enhancement.
Residual force enhancement is puzzling because it cannot be directly explained by the classical force–length relationship and the sliding filament theory of contraction, the main paradigms in the muscle field.
We used custom‐built instruments to measure residual force enhancement in skeletal myofibrils, and, for the first time, in cardiac myofibrils.
Our data report that residual force enhancement is present in skeletal muscles, but not cardiac muscles, and is regulated by the different isoforms of the titin protein filaments.
Abstract
When a skeletal muscle contracts isometrically, the muscle produces a force that is relative to the final isometric sarcomere length (SL). However, when the same final SL is reached by stretching the muscle while it contracts, the muscle produces a relatively higher force: a phenomenon commonly referred to as residual force enhancement. In this study, we investigated residual force enhancement in rabbit skeletal psoas myofibrils and, for the first time, cardiac papillary myofibrils. A custom‐built atomic force microscope was used in experiments that stretched myofibrils before and after inhibiting myosin and actin interactions to determine whether the different cardiac and skeletal titin isoforms regulate residual force enhancement. At SLs ranging from 2.24 to 3.13 μm, the skeletal myofibrils enhanced the force by an average of 9.0%, and by 29.5% after hindering myosin and actin interactions. At SLs ranging from 1.80 to 2.29 μm, the cardiac myofibrils did not enhance the force before or after hindering myosin and actin interactions. We conclude that residual force enhancement is present only in skeletal muscles and is dependent on the titin isoforms.

Key points
The early postnatal development of functional corticospinal connections in human infants is not fully clarified.
Corticospinal drive to upper and lower limb muscle shows developmental changes with an increased functional coupling in infants between 9 and 25 weeks in the beta frequency band.
The changes in functional coupling coincide with the developmental period where fidgety movements are present in healthy infants.
Data support a possible sensitive period where functional connections between corticospinal tract fibres and spinal motoneurones undergo activity‐dependent reorganization.
Abstract
The early postnatal development of functional corticospinal connections in human infants is not fully clarified. We used EEG and EMG to investigate the development of corticomuscular and intramuscular coherence as indicators of functional corticospinal connectivity in healthy infants aged 1–66 weeks. EEG was recorded over leg and hand area of motor cortex. EMG recordings were made from right ankle dorsiflexor and right wrist extensor muscles. Quantification of the amount of corticomuscular coherence in the 20–40 Hz frequency band showed a significantly larger coherence for infants aged 9–25 weeks compared to younger and older infants. Coherence between paired EMG recordings from tibialis anterior muscle in the 20–40 Hz frequency band was also significantly larger for the 9–25 week age group. A low‐amplitude, broad‐duration (40–50 ms) central peak of EMG–EMG synchronization was observed for infants younger than 9 weeks, whereas a short‐lasting (10–20 ms) central peak was observed for EMG–EMG synchronization in older infants. This peak was largest for infants aged 9–25 weeks. These data suggest that the corticospinal drive to lower and upper limb muscles shows significant developmental changes with an increase in functional coupling in infants aged 9–25 weeks, a period which coincides partly with the developmental period of normal fidgety movements. We propose that these neurophysiological findings may reflect the existence of a sensitive period where the functional connections between corticospinal tract fibres and spinal motoneurones undergo activity‐dependent reorganization. This may be relevant for the timing of early therapy interventions in infants with pre‐ and perinatal brain injury.

Key points
The present study suggests that the electrogenic sodium–bicarbonate cotransporter, NBCe1, supported by carbonic anhydrase II, CAII, provides an efficient mechanism of bicarbonate sensing in cortical astrocytes. This mechanism is proposed to play a major role in setting the pHi responses to extracellular acid/base challenges in astrocytes.
A decrease in extracellular [HCO3−] during isocapnic acidosis and isohydric hypocapnia, or an increase in intracellular [HCO3−] during hypercapnic acidosis, was effectively sensed by NBCe1, which carried bicarbonate out of the cells under these conditions, and caused an acidification and sodium fall in WT astrocytes, but not in NBCe1‐knockout astrocytes.
Isocapnic acidosis, hypercapnic acidosis and isohydric hypocapnia evoked inward currents in NBCe1‐ and CAII‐expressing Xenopus laevis oocytes, but not in native oocytes, suggesting that NBCe1 operates in the outwardly directed mode under these conditions consistent with our findings in astrocytes.
We propose that bicarbonate sensing of astrocytes may have functional significance during extracellular acid/base disturbances in the brain, as it not only alters intracellular pH/[HCO3−]‐dependent functions of astrocytes, but also modulates the extracellular pH/[HCO3−] in brain tissue.
Abstract
Extracellular acid/base status of the mammalian brain undergoes dynamic changes during many physiological and pathological events. Although intracellular pH (pHi) of astrocytes responds to extracellular acid/base changes, the mechanisms mediating these changes have remained unresolved. We have previously shown that the electrogenic sodium–bicarbonate cotransporter, NBCe1, is a high‐affinity bicarbonate carrier in cortical astrocytes. In the present study, we investigated whether NBCe1 plays a role in bicarbonate sensing in astrocytes, and in determining the pHi responses to extracellular acid/base challenges. We measured changes in intracellular H+ and Na+ in astrocytes from wild‐type (WT) and from NBCe1‐knockout (KO) mice, using ion‐selective dyes, during isocapnic acidosis, hypercapnic acidosis and hypocapnia. We also analysed NBCe1‐mediated membrane currents in Xenopus laevis oocytes under similar conditions. Comparing WT and NBCe1‐KO astrocytes, we could dissect the contribution of NBCe1, of diffusion of CO2 across the cell membrane and, after blocking carbonic anhydrase (CA) activity with ethoxyzolamide, of the role of CA, for the amplitude and rate of acid/base fluxes. Our results suggest that NBCe1 transport activity in astrocytes, supported by CA activity, renders astrocytes bicarbonate sensors in the mouse cortex. NBCe1 carried bicarbonate into and out of the cell by sensing the variations of transmembrane [HCO3−], irrespective of the changes in intra‐ and extracellular pH, and played a major role in setting pHi responses to the extracellular acid/base challenges. We propose that bicarbonate sensing of astrocytes may have potential functional significance during extracellular acid/base alterations in the brain.

Key Points
Astronauts have recently been discovered to have impaired vision, with a presentation that resembles syndromes of elevated intracranial pressure on Earth.
Gravity has a profound effect on fluid distribution and pressure within the human circulation. In contrast to prevailing theory, we observed that microgravity reduces central venous and intracranial pressure.
This being said, intracranial pressure is not reduced to the levels observed in the 90 deg seated upright posture on Earth. Thus, over 24 h in zero gravity, pressure in the brain is slightly above that observed on Earth, which may explain remodelling of the eye in astronauts.
Abstract
Astronauts have recently been discovered to have impaired vision, with a presentation that resembles syndromes of elevated intracranial pressure (ICP). This syndrome is considered the most mission‐critical medical problem identified in the past decade of manned spaceflight. We recruited five men and three women who had an Ommaya reservoir inserted for the delivery of prophylactic CNS chemotherapy, but were free of their malignant disease for at least 1 year. ICP was assessed by placing a fluid‐filled 25 gauge butterfly needle into the Ommaya reservoir. Subjects were studied in the upright and supine position, during acute zero gravity (parabolic flight) and prolonged simulated microgravity (6 deg head‐down tilt bedrest). ICP was lower when seated in the 90 deg upright posture compared to lying supine (seated, 4 ± 1 vs. supine, 15 ± 2 mmHg). Whilst lying in the supine posture, central venous pressure (supine, 7 ± 3 vs. microgravity, 4 ± 2 mmHg) and ICP (supine, 17 ± 2 vs. microgravity, 13 ± 2 mmHg) were reduced in acute zero gravity, although not to the levels observed in the 90 deg seated upright posture on Earth. Prolonged periods of simulated microgravity did not cause progressive elevations in ICP (supine, 15 ± 2 vs. 24 h head‐down tilt, 15 ± 4 mmHg). Complete removal of gravity does not pathologically elevate ICP but does prevent the normal lowering of ICP when upright. These findings suggest the human brain is protected by the daily circadian cycles in regional ICPs, without which pathology may occur.

Key points
Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell‐to‐cell diffusion of ions, metabolites and second messengers.
Stimulation of the adenosine receptor subtype A2B increases the gap junction coupling in the human blood–brain barrier endothelial cell line hCMEC/D3.
Although the increased gap junction coupling is cAMP‐dependent, neither the protein kinase A nor the exchange protein directly activated by cAMP were involved in this increase.
We found that cAMP activates cyclic nucleotide‐gated (CNG) channels and thereby induces a Ca2+ influx, which leads to the increase in gap junction coupling.
The report identifies CNG channels as a possible physiological link between adenosine receptors and the regulation of gap junction channels in endothelial cells of the blood–brain barrier.
Abstract
The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood–brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT‐PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2A and A2B adenosine receptor subtype agonist 2‐phenylaminoadenosine (2‐PAA) on the gap junction coupling. We found that 2‐PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration‐dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2‐PAA‐related enhancement of gap junction coupling. In contrast, the cyclic nucleotide‐gated (CNG) channel inhibitor l‐cis‐diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+, suppressed the 2‐PAA‐related enhancement of gap junction coupling. Moreover, we observed a 2‐PAA‐dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP‐dependent manner. Ca2+ in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor‐dependent signalling of endothelial cells of the blood–brain barrier.

Key points
The internal anal sphincter develops tone important for maintaining high anal pressure and continence. Controversy exists regarding the mechanisms underlying tone development.
We examined the hypothesis that tone depends upon electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC‐IM) by activation of Ca2+‐activated Cl− channels (ANO1, encoded by Ano1) and voltage‐dependent L‐type Ca2+ channels (CavL, encoded by Cacna1c).
Measurement of membrane potential and contraction indicated that ANO1 and CavL have a central role in SW generation, phasic contractions and tone, independent of stretch.
ANO1 expression was examined in wildtype and Ano1/+egfp mice with immunohistochemical techniques. Ano1 and Cacna1c expression levels were examined by quantitative PCR in fluorescence‐activated cell sorting.
ICC‐IM were the predominant cell type expressing ANO1 and the most likely candidate for SW generation. SWs in ICC‐IM are proposed to conduct to smooth muscle where Ca2+ entry via CavL results in phasic activity that sums to produce tone.
Abstract
The mechanism underlying tone generation in the internal anal sphincter (IAS) is controversial. We examined the hypothesis that tone depends upon generation of electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC‐IM) by activation of Ca2+‐activated Cl− channels (encoded by Ano1) and voltage‐dependent L‐type Ca2+ channels (encoded by Cacna1c). Phasic contractions and tone in the IAS were nearly abolished by ANO1 and CavL antagonists. ANO1 antagonists also abolished SWs as well as transient depolarizations that persisted after addition of CavL antagonists. Tone development in the IAS did not require stretch of muscles, and the sensitivity of contraction to ANO1 antagonists was the same in stretched versus un‐stretched muscles. ANO1 expression was examined in wildtype and Ano1/+egfp mice with immunohistochemical techniques. Dual labelling revealed that ANO1 expression could be resolved in ICC but not smooth muscle cells (SMCs) in the IAS and rectum. Ano1, Cacna1c and Kit gene expression were the same in extracts of IAS and rectum muscles. In IAS cells isolated with fluorescence‐activated cell sorting, Ano1 expression was 26.5‐fold greater in ICC than in SMCs while Cacna1c expression was only 2‐fold greater in SMCs than in ICC. These data support a central role for ANO1 and CavL in the generation of SWs and tone in the IAS. ICC‐IM are the probable cellular candidate for ANO1 currents and SW generation. We propose that ANO1 and CavL collaborate to generate SWs in ICC‐IM followed by conduction to adjacent SMCs where phasic calcium entry through CavL sums to produce tone.

Key points
Lung aeration at birth significantly increases pulmonary blood flow, which is unrelated to increased oxygenation or other spatial relationships that match ventilation to perfusion.
Using simultaneous X‐ray imaging and angiography in near‐term rabbits, we investigated the relative contributions of the vagus nerve and oxygenation to the increase in pulmonary blood flow at birth.
Vagal denervation inhibited the global increase in pulmonary blood flow induced by partial lung aeration, although high inspired oxygen concentrations can partially mitigate this effect.
The results of the present study indicate that a vagal reflex may mediate a rapid global increase in pulmonary blood flow in response to partial lung aeration.
Abstract
Air entry into the lungs at birth triggers major cardiovascular changes, including a large increase in pulmonary blood flow (PBF) that is not spatially related to regional lung aeration. To investigate the possible underlying role of a vagally‐mediated stimulus, we used simultaneous phase‐contrast X‐ray imaging and angiography in near‐term (30 days of gestation) vagotomized (n = 15) or sham‐operated (n = 15) rabbit kittens. Rabbits were imaged before ventilation, when one lung was ventilated (unilateral) with 100% nitrogen (N2), air or 100% oxygen (O2), and after all kittens were switched to unilateral ventilation in air and then ventilation of both lungs using air. Compared to control kittens, vagotomized kittens had little or no increase in PBF in both lungs following unilateral ventilation when ventilation occurred with 100% N2 or with air. However, relative PBF did increase in vagotomized animals ventilated with 100% O2, indicating the independent stimulatory effects of local oxygen concentration and autonomic innervation on the changes in PBF at birth. These findings demonstrate that vagal denervation inhibits the previously observed increase in PBF with partial lung aeration, although high inspired oxygen concentrations can partially mitigate this effect.

Key points
The ductal system of the pancreas secretes large volumes of alkaline fluid containing HCO3− concentrations as high as 140 mm during hormonal stimulation.
A computational model has been constructed to explore the underlying ion transport mechanisms. Parameters were estimated by fitting the model to experimental data from guinea‐pig pancreatic ducts.
The model was readily able to secrete 140 mm HCO3−. Its capacity to do so was not dependent upon special properties of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channels and solute carrier family 26 member A6 (SLC26A6) anion exchangers.
We conclude that the main requirement for secreting high HCO3− concentrations is to minimize the secretion of Cl− ions.
These findings help to clarify the mechanism responsible for pancreatic HCO3− secretion, a vital process that prevents the formation of protein plugs and viscous mucus in the ducts, which could otherwise lead to pancreatic disease.
Abstract
A computational model of guinea‐pig pancreatic duct epithelium was developed to determine the transport mechanism by which HCO3− ions are secreted at concentrations in excess of 140 mm. Parameters defining the contributions of the individual ion channels and transporters were estimated by least‐squares fitting of the model predictions to experimental data obtained from isolated ducts and intact pancreas under a range of experimental conditions. The effects of cAMP‐stimulated secretion were well replicated by increasing the activities of the basolateral Na+‐HCO3− cotransporter (NBC1) and apical Cl−/HCO3− exchanger (solute carrier family 26 member A6; SLC26A6), increasing the basolateral K+ permeability and apical Cl− and HCO3− permeabilities (CFTR), and reducing the activity of the basolateral Cl−/HCO3− exchanger (anion exchanger 2; AE2). Under these conditions, the model secreted ∼140 mm HCO3− at a rate of ∼3 nl min−1 mm−2, which is consistent with experimental observations. Alternative 1:2 and 1:1 stoichiometries for Cl−/HCO3− exchange via SLC26A6 at the apical membrane were able to support a HCO3−‐rich secretion. Raising the HCO3−/Cl− permeability ratio of CFTR from 0.4 to 1.0 had little impact upon either the secreted HCO3− concentration or the volume flow. However, modelling showed that a reduction in basolateral AE2 activity by ∼80% was essential in minimizing the intracellular Cl− concentration following cAMP stimulation and thereby maximizing the secreted HCO3− concentration. The addition of a basolateral Na+‐K+‐2Cl− cotransporter (NKCC1), assumed to be present in rat and mouse ducts, raised intracellular Cl− and resulted in a lower secreted HCO3− concentration, as is characteristic of those species. We conclude therefore that minimizing the driving force for Cl− secretion is the main requirement for secreting 140 mm HCO3−.

Key points
A high‐fat diet (60% kcal from fat) is associated with motility disorders inducing constipation and loss of nitrergic myenteric neurons in the proximal colon. Gut microbiota dysbiosis, which occurs in response to HFD, contributes to endotoxaemia. High levels of lipopolysaccharide lead to apoptosis in cultured myenteric neurons that express Toll‐like receptor 4 (TLR4).
Consumption of a Western diet (WD) (35% kcal from fat) for 6 weeks leads to gut microbiota dysbiosis associated with altered bacterial metabolites and increased levels of plasma free fatty acids. These disorders precede the nitrergic myenteric cell loss observed in the proximal colon.
Mice lacking TLR4 did not exhibit WD‐induced myenteric cell loss and dysmotility. Lipopolysaccharide‐induced in vitro enteric neurodegeneration requires the presence of palmitate and may be a result of enhanced NO production.
The present study highlights the critical role of plasma saturated free fatty acids that are abundant in the WD with respect to driving enteric neuropathy and colonic dysmotility.
Abstract
The consumption of a high‐fat diet (HFD) is associated with myenteric neurodegeneration, which in turn is associated with delayed colonic transit and constipation. We examined the hypothesis that an inherent increase in plasma free fatty acids (FFA) in the HFD together with an HFD‐induced alteration in gut microbiota contributes to the pathophysiology of these disorders. C57BL/6 mice were fed a Western diet (WD) (35% kcal from fat enriched in palmitate) or a purified regular diet (16.9% kcal from fat) for 3, 6, 9 and 12 weeks. Gut microbiota dysbiosis was investigated by fecal lipopolysaccharide (LPS) measurement and metabolomics (linear trap quadrupole‐Fourier transform mass spectrometer) analysis. Plasma FFA and LPS levels were assessed, in addition to colonic and ileal nitrergic myenteric neuron quantifications and motility. Compared to regular diet‐fed control mice, WD‐fed mice gained significantly more weight without blood glucose alteration. Dysbiosis was exhibited after 6 weeks of feeding, as reflected by increased fecal LPS and bacterial metabolites and concomitant higher plasma FFA. The numbers of nitrergic myenteric neurons were reduced in the proximal colon after 9 and 12 weeks of WD and this was also associated with delayed colonic transit. WD‐fed Toll‐like receptor 4 (TLR4)−/− mice did not exhibit myenteric cell loss or dysmotility. Finally, LPS (0.5–2 ng·ml–1) and palmitate (20 and 30 μm) acted synergistically to induce neuronal cell death in vitro, which was prevented by the nitric oxide synthase inhibitor NG‐nitro‐l‐arginine methyl ester. In conclusion, WD‐feeding results in increased levels of FFA and microbiota that, even in absence of hyperglycaemia or overt endotoxaemia, synergistically induce TLR4‐mediated neurodegeneration and dysmotility.

Key points
Older pregnant women have a greater risk of operative delivery, still birth and post‐term induction.
This suggests that maternal age can influence the timing of birth and processes of parturition.
We have found that increasing maternal age in C57BL/6J mice is associated with prolongation of gestation and length of labour.
Older pregnant mice also had delayed progesterone withdrawal and impaired myometrial function.
Uterine ageing and labour dysfunction should be investigated further in older primigravid women.
Abstract
Advanced maternal age (≥35 years) is associated with increased rates of operative delivery, stillbirth and post‐term labour induction. The physiological causes remain uncertain, although impaired myometrial function has been implicated. To investigate the hypothesis that maternal age directly influences successful parturition, we assessed the timing of birth and fetal outcome in pregnant C57BL/6J mice at 3 months (young) and 5 months (intermediate) vs. 8 months (older) of age using infrared video recording. Serum progesterone profiles, myometrium and cervix function, and mitochondrial electron transport chain complex enzymatic activities were also examined. Older pregnant mice had a longer mean gestation and labour duration (P

Key points
N‐cadherin formed punctate adherens junctions (AJ) along the borders between vascular smooth muscle cells (VSMCs) in the pressurized rat superior cerebellar artery.
The formation of N‐cadherin AJs in the vessel wall depends on the intraluminal pressure and was responsive to treatment with phenylephrine (PE) (10−5 m) and ACh (10−5 m).
N‐cadherin‐coated beads were able to induce clustering of N‐cadherin‐enhanced green fluorescent protein (EGFP) on the plasma membrane of isolated VSMCs, whereas treatment with PE (10−5 m) or sodium nitroprusside (10−5 m) induced a significant increase or decrease in the N‐cadherin‐EGFP clustering, respectively.
Application of pulling force (∼1 nN) to the N‐cadherin‐coated beads via an atomic force microscope induced a localized mechanical response from the VSMCs that opposed the pulling.
Abstract
N‐cadherin is the major cell–cell adhesion molecule in vascular smooth muscle cells (VSMCs). We tested the hypothesis that N‐cadherin is part of a novel mechanosensory mechanism in VSMCs and plays an active role in both the arteriolar myogenic response and during changes in vascular tone induced by vasomotor agonists. Intact and pressurized rat superior cerebellar arteries were labelled for confocal immunofluorescence imaging. N‐cadherin formed punctate adherens junctions (AJ) along the borders between VSMCs. When the lumen pressure was raised from 50 to 90 mmHg, both the density and the average size of N‐cadherin AJs increased significantly. Similarly, arteriolar constriction with phenylephrine (PE) (10–5 m) induced a significant increase of N‐cadherin AJ density at 50 mmHg, whereas vasodilatation induced by ACh (10–5 m) was accompanied by a significant decrease in density and size of N‐cadherin AJs. An atomic force microscope (AFM) was employed to further examine the mechano‐responsive properties of N‐cadherin adhesion sites in isolated VSMCs. AFM probes with an attached N‐cadherin‐coated microbead (5 μm) induced a progressive clustering of N‐cadherin‐enhanced green fluorescent protein (EGFP) on the VSMC surface. Application of pulling force (∼1 nN) to the N‐cadherin‐coated‐beads with the AFM induced a localized mechanical response from the VSMCs that opposed the pulling. Treatment with PE (10–5 m) or sodium nitroprusside (10–5 m) induced a significant increase or decrease of the N‐cadherin‐EGFP clustering, respectively. These observations provide compelling evidence that N‐cadherin AJs are sensitive to pressure and vasomotor agonists in VSMCs and support a functional role of N‐cadherin AJs in vasomotor regulation.

Key points
Upon repeated application of short ACh pulses to C57BL6J mouse chromaffin cells, the amperometrically monitored secretory responses promptly decayed to a steady‐state level of around 25% of the initial response. A subsequent K+ pulse, however, overcame such decay. These data suggest that mouse chromaffin cells have a ready release‐vesicle pool that is selectively recruited by the physiological neurotransmitter ACh.
The ACh‐sensitive vesicle pool is refilled and maintained by the rate of Ca2+ delivery from mitochondria to the cytosol, through the mitochondrial Na+/Ca2+ exchanger (mNCX). ITH12662, a novel blocker of the mNCX, prevented the decay of secretion elicited by ACh pulses and delayed the rate of [Ca2+]c clearance.
This regulatory pathway may be physiologically relevant in situations of prolonged stressful conflicts where a sustained catecholamine release is regulated by mitochondrial Ca2+ circulation through the mNCX, which couples respiration and ATP synthesis to long‐term stimulation of chromaffin cells by endogenously released ACh.
Abstract
Using caged‐Ca2+ photorelease or paired depolarising pulses in voltage‐clamped chromaffin cells (CCs), various pools of secretory vesicles with different readiness to undergo exocytosis have been identified. Whether these pools are present in unclamped CCs challenged with ACh, the physiological neurotransmitter at the splanchnic nerve‐CC synapse, is unknown. We have explored here whether an ACh‐sensitive ready‐release vesicle pool (ASP) is present in C57BL6J mouse chromaffin cells (MCCs). Single cells were fast perfused with a Tyrode solution at 37°C, and challenged with 12 sequential ACh pulses (100 μm, 2 s, every 30 s) plus a K+ pulse given at the end (75 mm K+). After the first 2–3 ACh pulses the amperometrically monitored secretory responses promptly decayed to a steady‐state level of around 25% of the initial response. The last K+ pulse, however, overcame such decay. Repeated ACh pulses to voltage‐clamped cells elicited non‐desensitising nicotinic currents. Also, the [Ca2+]c transients elicited by repeated ACh pulses that were superimposed on a stable baseline elevation did not undergo decay. The novel blocker of the mitochondrial Na+/Ca2+ exchanger (mNCX) ITH12662 prevented the decay of secretion elicited by ACh pulses and delayed the rate of [Ca2+]c clearance. The experiments are compatible with the idea that C57BL6J MCCs have an ASP vesicle pool that is selectively recruited by the physiological neurotransmitter ACh and is regulated by the rate of Ca2+ delivery from mitochondria to the cytosol, through the mNCX.

Key points
Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac illness and can lead to diastolic dysfunction, sudden cardiac death and heart failure.
Treatment of HCM patients is empirical and current pharmacological treatments are unable to stop disease progression or reverse hypertrophy.
In this study, we tested if the non‐dihydropyridine Ca2+ channel blocker diltiazem, which previously showed potential to stop disease progression, can improve the phenotype of a HCM mouse model (Mybpc3‐targeted knock‐in), which is based on a mutation commonly found in patients.
Diltiazem improved contractile function of isolated ventricular cardiomyocytes acutely, but chronic application did not improve the phenotype of adult mice with a fully developed HCM.
Our study shows that diltiazem has beneficial effects in HCM, but long‐term treatment success is likely to depend on characteristics and cause of HCM and onset of treatment.
Abstract
Left ventricular hypertrophy, diastolic dysfunction and fibrosis are the main features of hypertrophic cardiomyopathy (HCM). Guidelines recommend β‐adrenoceptor or Ca2+ channel antagonists as pharmacological treatment. The Ca2+ channel blocker diltiazem recently showed promising beneficial effects in pre‐clinical HCM, particularly in patients carrying MYBPC3 mutations. In the present study we evaluated whether diltiazem could ameliorate or reverse the disease phenotype in cells and in vivo in an Mybpc3‐targeted knock‐in (KI) mouse model of HCM. Sarcomere shortening and Ca2+ transients were measured in KI and wild‐type (WT) cardiomyocytes in basal conditions (1‐Hz pacing) and under stress conditions (30 nm isoprenaline, 5‐Hz pacing) with or without pre‐treatment with 1 μm diltiazem. KI cardiomyocytes exhibited lower diastolic sarcomere length (dSL) at baseline, a tendency to a stronger positive inotropic response to isoprenaline than WT, a marked reduction of dSL and a tendency towards arrhythmias under stress conditions. Pre‐treatment of cardiomyocytes with 1 μm diltiazem reduced the drop in dSL and arrhythmia frequency in KI, and attenuated the positive inotropic effect of isoprenaline. Furthermore, diltiazem reduced the contraction amplitude at 5 Hz but did not affect diastolic Ca2+ load and Ca2+ transient amplitude. Six months of diltiazem treatment of KI mice did not reverse cardiac hypertrophy and dysfunction, activation of the fetal gene program or fibrosis. In conclusion, diltiazem blunted the response to isoprenaline in WT and KI cardiomyocytes and improved diastolic relaxation under stress conditions in KI cardiomyocytes. This beneficial effect of diltiazem in cells did not translate in therapeutic efficacy when applied chronically in KI mice.

Key points
Muscle‐derived thermogenesis during acute cold exposure in humans consists of a combination of cold‐induced increases in skeletal muscle proton leak and shivering.
Daily cold exposure results in an increase in brown adipose tissue oxidative capacity coupled with a decrease in the cold‐induced skeletal muscle proton leak and shivering intensity.
Improved coupling between electromyography‐determined muscle activity and whole‐body heat production following cold acclimation suggests a maintenance of ATPase‐dependent thermogenesis and decrease in skeletal muscle ATPase independent thermogenesis.
Although daily cold exposure did not change the fibre composition of the vastus lateralis, the fibre composition was a strong predictor of the shivering pattern evoked during acute cold exposure.
Abstract
We previously showed that 4 weeks of daily cold exposure in humans can increase brown adipose tissue (BAT) volume by 45% and oxidative metabolism by 182%. Surprisingly, we did not find a reciprocal reduction in shivering intensity when exposed to a mild cold (18°C). The present study aimed to determine whether changes in skeletal muscle oxidative metabolism or shivering activity could account for these unexpected findings. Nine men participated in a 4 week cold acclimation intervention (10°C water circulating in liquid‐conditioned suit, 2 h day–1, 5 days week–1). Shivering intensity and pattern were measured continuously during controlled cold exposure (150 min at 4 °C) before and after the acclimation. Muscle biopsies from the m. vastus lateralis were obtained to measure oxygen consumption rate and proton leak of permeabilized muscle fibres. Cold acclimation elicited a modest 21% (P

Key points
Combining nitric oxide (NO)‐mediated increased blood flow with angiopoietin‐1–Tie2 receptor signalling induces arteriolargenesis – the formation of arterioles from capillaries – in a model of physiological angiogenesis.
This NO–Tie‐mediated arteriolargenesis requires endogenous vascular endothelial growth factor (VEGF) signalling.
Inhibition of VEGF signalling increases pericyte coverage in microvessels.
Together these findings indicate that generation of functional neovasculature requires close titration of NO–Tie2 signalling and localized VEGF induction, suggesting that the use of exogenous VEGF expression as a therapeutic for neovascularization may not be successful.
Abstract
Signalling through vascular endothelial growth factor (VEGF) receptors and the tyrosine kinase with IgG and EGF domains‐2 (Tie2) receptor by angiopoietins is required in combination with blood flow for the formation of a functional vascular network. We tested the hypothesis that VEGF and angiopoietin‐1 (Ang1) contribute differentially to neovascularization induced by nitric oxide (NO)‐mediated vasodilatation, by comparing the phenotype of new microvessels in the mesentery during induction of vascular remodelling by over‐expression of endothelial nitric oxide synthase in the fat pad of the adult rat mesentery during inhibition of angiopoietin signalling with soluble Tie2 (sTie2) and VEGF signalling with soluble Fms‐like tyrosine kinase receptor‐1 (sFlt1). We found that NO‐mediated angiogenesis was blocked by inhibition of VEGF with sFlt1 (from 881 ± 98% increase in functional vessel area to 279 ± 72%) and by inhibition of angiopoietin with sTie2 (to 337 ± 67%). Exogenous angiopoietin‐1 was required to induce arteriolargenesis (8.6 ± 1.3% of vessels with recruitment of vascular smooth muscle cells; VSMCs) in the presence of enhanced flow. sTie2 and sFlt1 both inhibited VSMC recruitment (both 0%), and VEGF inhibition increased pericyte recruitment to newly formed vessels (from 27 ± 2 to 54 ± 3% pericyte ensheathment). We demonstrate that a fine balance of VEGF and angiopoietin signalling is required for the formation of a functional vascular network. Endogenous VEGF signalling prevents excess neovessel pericyte coverage, and is required for VSMC recruitment during increased nitric oxide‐mediated vasodilatation and angiopoietin signalling (NO–Tie‐mediated arteriogenesis). Therapeutic vascular remodelling paradigms may therefore require treatments that modulate blood flow to utilize endogenous VEGF, in combination with exogenous Ang1, for effective neovascularization.

Key points
Just as a portrait painting can come from a collection of coarse and fine details, natural vision can be decomposed into coarse and fine components.
Previous studies have shown that the early visual areas in the brain represent these components in a map‐like fashion.
Other studies have shown that these same visual areas can be sensitive to how coarse and fine features line up in space.
We found that the brain actually jointly represents both the scale of the feature (fine, medium, or coarse) and the alignment of these features in space.
The results suggest that the visual cortex has an optimized representation particularly for the alignment of fine details, which are crucial in understanding the visual scene.
Abstract
Complex natural scenes can be decomposed into their oriented spatial frequency (SF) and phase relationships, both of which are represented locally at the earliest stages of cortical visual processing. The SF preference map in the human cortex, obtained using synthetic stimuli, is orderly and correlates strongly with eccentricity. In addition, early visual areas show sensitivity to the phase information that describes the relationship between SFs and thereby dictates the structure of the image. Taken together, two possibilities arise for the joint representation of SF and phase: either the entirety of the cortical SF map is uniformly sensitive to phase, or a particular set of SFs is selectively phase sensitive – for example, greater phase sensitivity for higher SFs that define fine‐scale edges in a complex scene. To test between these two possibilities, we constructed a novel continuous natural scene video whereby phase information was maintained in one SF band but scrambled elsewhere. By shifting the central frequency of the phase‐aligned band in time, we mapped the phase‐sensitive SF preference of the visual cortex. Using functional magnetic resonance imaging, we found that phase sensitivity in early visual areas is biased toward higher SFs. Compared to a SF map of the same scene obtained using linear‐filtered stimuli, a much larger patch of areas V1 and V2 is sensitive to the phase alignment of higher SFs. The results of early areas cannot be explained by attention. Our results suggest non‐uniform sensitivity to phase alignment in population‐level SF representations, with phase alignment being particularly important for fine‐scale edge representations of natural scenes.

Key points
Patients with diabetes show a blunted cardiac inotropic response to β‐adrenergic stimulation despite normal cardiac contractile reserve.
Acute insulin stimulation impairs β‐adrenergically induced contractile function in isolated cardiomyocytes and Langendorff‐perfused hearts.
In this study, we aimed to examine the potential effects of hyperinsulinaemia associated with high‐fat diet (HFD) feeding on the cardiac β2‐adrenergic receptor signalling and the impacts on cardiac contractile function.
We showed that 8 weeks of HFD feeding leads to reductions in cardiac functional reserve in response to β‐adrenergic stimulation without significant alteration of cardiac structure and function, which is associated with significant changes in β2‐adrenergic receptor phosphorylation at protein kinase A and G‐protein receptor kinase sites in the myocardium.
The results suggest that clinical intervention might be applied to subjects in early diabetes without cardiac symptoms to prevent further cardiac complications.
Abstract
Patients with diabetes display reduced exercise capability and impaired cardiac contractile reserve in response to adrenergic stimulation. We have recently uncovered an insulin receptor and adrenergic receptor signal network in the heart. The aim of this study was to understand the impacts of high‐fat diet (HFD) on the insulin–adrenergic receptor signal network in hearts. After 8 weeks of HFD feeding, mice exhibited diabetes, with elevated insulin and glucose concentrations associated with body weight gain. Mice fed an HFD had normal cardiac structure and function. However, the HFD‐fed mice displayed a significant elevation of phosphorylation of the β2‐adrenergic receptor (β2AR) at both the protein kinase A site serine 261/262 and the G‐protein‐coupled receptor kinase site serine 355/356 and impaired adrenergic reserve when compared with mice fed on normal chow. Isolated myocytes from HFD‐fed mice also displayed a reduced contractile response to adrenergic stimulation when compared with those of control mice fed normal chow. Genetic deletion of the β2AR led to a normalized adrenergic response and preserved cardiac contractile reserve in HFD‐fed mice. Together, these data indicate that HFD promotes phosphorylation of the β2AR, contributing to impairment of cardiac contractile reserve before cardiac structural and functional remodelling, suggesting that early intervention in the insulin–adrenergic signalling network might be effective in prevention of cardiac complications in diabetes.

Key points
We tested perceived head‐on‐feet orientation and the direction of vestibular‐evoked balance responses in passively and actively held head‐turned postures.
The direction of vestibular‐evoked balance responses was not aligned with perceived head‐on‐feet orientation while maintaining prolonged passively held head‐turned postures. Furthermore, static visual cues of head‐on‐feet orientation did not update the estimate of head posture for the balance controller.
A prolonged actively held head‐turned posture did not elicit a rotation in the direction of the vestibular‐evoked balance response despite a significant rotation in perceived angular head posture.
It is proposed that conscious perception of head posture and the transformation of vestibular signals for standing balance relying on this head posture are not dependent on the same internal representation. Rather, the balance system may operate under its own sensorimotor principles, which are partly independent from perception.
Abstract
Vestibular signals used for balance control must be integrated with other sensorimotor cues to allow transformation of descending signals according to an internal representation of body configuration. We explored two alternative models of sensorimotor integration that propose (1) a single internal representation of head‐on‐feet orientation is responsible for perceived postural orientation and standing balance or (2) conscious perception and balance control are driven by separate internal representations. During three experiments, participants stood quietly while passively or actively maintaining a prolonged head‐turned posture (>10 min). Throughout the trials, participants intermittently reported their perceived head angular position, and subsequently electrical vestibular stimuli were delivered to elicit whole‐body balance responses. Visual recalibration of head‐on‐feet posture was used to determine whether static visual cues are used to update the internal representation of body configuration for perceived orientation and standing balance. All three experiments involved situations in which the vestibular‐evoked balance response was not orthogonal to perceived head‐on‐feet orientation, regardless of the visual information provided. For prolonged head‐turned postures, balance responses consistent with actual head‐on‐feet posture occurred only during the active condition. Our results indicate that conscious perception of head‐on‐feet posture and vestibular control of balance do not rely on the same internal representation, but instead treat sensorimotor cues in parallel and may arrive at different conclusions regarding head‐on‐feet posture. The balance system appears to bypass static visual cues of postural orientation and mainly use other sensorimotor signals of head‐on‐feet position to transform vestibular signals of head motion, a mechanism appropriate for most daily activities.

Key points
The intracellular concentration of free calcium ions ([Ca2+]i) in a nerve terminal controls both transmitter release and synaptic plasticity.
The rapid triggering of transmitter release depends on the local micro‐ or nanodomain of highly elevated [Ca2+]i in the vicinity of open voltage‐gated Ca2+ channels, whereas short‐term synaptic plasticity is often controlled by global changes in residual [Ca2+]i, averaged over the whole nerve terminal volume.
Here we describe dynamic changes of such global [Ca2+]i in the calyx of Held – a giant mammalian glutamatergic nerve terminal, which is particularly suited for biophysical studies.
We provide quantitative data on Ca2+ inflow, Ca2+ buffering and Ca2+ clearance.
These data allow us to predict changes in [Ca2+]i in the nerve terminal in response to a wide range of stimulus protocols at high temporal resolution and provide a basis for the modelling of short‐term plasticity of glutamatergic synapses.
Abstract
Many aspects of short‐term synaptic plasticity (STP) are controlled by relatively slow changes in the presynaptic intracellular concentration of free calcium ions ([Ca2+]i) that occur in the time range of a few milliseconds to several seconds. In nerve terminals, [Ca2+]i equilibrates diffusionally during such slow changes, such that the globally measured, residual [Ca2+]i that persists after the collapse of local domains is often the appropriate parameter governing STP. Here, we study activity‐dependent dynamic changes in global [Ca2+]i at the rat calyx of Held nerve terminal in acute brainstem slices using patch‐clamp and microfluorimetry. We use low concentrations of a low‐affinity Ca2+ indicator dye (100 μm Fura‐6F) in order not to overwhelm endogenous Ca2+ buffers. We first study voltage‐clamped terminals, dialysed with pipette solutions containing minimal amounts of Ca2+ buffers, to determine Ca2+ binding properties of endogenous fixed buffers as well as the mechanisms of Ca2+ clearance. Subsequently, we use pipette solutions including 500 μm EGTA to determine the Ca2+ binding kinetics of this chelator. We provide a formalism and parameters that allow us to predict [Ca2+]i changes in calyx nerve terminals in response to a wide range of stimulus protocols. Unexpectedly, the Ca2+ affinity of EGTA under the conditions of our measurements was substantially lower (KD = 543 ± 51 nm) than measured in vitro, mainly as a consequence of a higher than previously assumed dissociation rate constant (2.38 ± 0.20 s−1), which we need to postulate in order to model the measured presynaptic [Ca2+]i transients.

Key points
We develop a new biophysically rooted, physiologically constrained conductance‐based synaptic model to mechanistically account for short‐term facilitation and depression, respectively through residual calcium and transmitter depletion kinetics.
We address the specific question of how presynaptic components (including voltage‐gated ion channels, pumps, buffers and release‐handling mechanisms) and interactions among them define synaptic filtering and short‐term plasticity profiles.
Employing global sensitivity analyses (GSAs), we show that near‐identical synaptic filters and short‐term plasticity profiles could emerge from disparate presynaptic parametric combinations with weak pairwise correlations.
Using virtual knockout models, a technique to address the question of channel‐specific contributions within the GSA framework, we unveil the differential and variable impact of each ion channel on synaptic physiology.
Our conclusions strengthen the argument that parametric and interactional complexity in biological systems should not be viewed from the limited curse‐of‐dimensionality standpoint, but from the evolutionarily advantageous perspective of providing functional robustness through degeneracy.
Abstract
Information processing in neurons is known to emerge as a gestalt of pre‐ and post‐synaptic filtering. However, the impact of presynaptic mechanisms on synaptic filters has not been quantitatively assessed. Here, we developed a biophysically rooted, conductance‐based model synapse that was endowed with six different voltage‐gated ion channels, calcium pumps, calcium buffer and neurotransmitter‐replenishment mechanisms in the presynaptic terminal. We tuned our model to match the short‐term plasticity profile and band‐pass structure of Schaffer collateral synapses, and performed sensitivity analyses to demonstrate that presynaptic voltage‐gated ion channels regulated synaptic filters through changes in excitability and associated calcium influx. These sensitivity analyses also revealed that calcium‐ and release‐control mechanisms were effective regulators of synaptic filters, but accomplished this without changes in terminal excitability or calcium influx. Next, to perform global sensitivity analysis, we generated 7000 randomized models spanning 15 presynaptic parameters, and computed eight different physiological measurements in each of these models. We validated these models by applying experimentally obtained bounds on their measurements, and found 104 (∼1.5%) models to match the validation criteria for all eight measurements. Analysing these valid models, we demonstrate that analogous synaptic filters emerge from disparate combinations of presynaptic parameters exhibiting weak pairwise correlations. Finally, using virtual knockout models, we establish the variable and differential impact of different presynaptic channels on synaptic filters, underlining the critical importance of interactions among different presynaptic components in defining synaptic physiology. Our results have significant implications for protein‐localization strategies required for physiological robustness and for degeneracy in long‐term synaptic plasticity profiles.

Key points
In atrial myocytes excitation–contraction coupling is strikingly different from ventricle because atrial myocytes lack a transverse tubule membrane system: Ca2+ release starts in the cell periphery and propagates towards the cell centre by Ca2+‐induced Ca2+ release from the sarcoplasmic reticulum (SR) Ca2+ store.
The cytosolic Ca2+ sensitivity of the ryanodine receptor (RyRs) Ca2+ release channel is low and it is unclear how Ca2+ release can be activated in the interior of atrial cells.
Simultaneous confocal imaging of cytosolic and intra‐SR calcium revealed a transient elevation of store Ca2+ that we termed ‘Ca2+ sensitization signal’.
We propose a novel paradigm of atrial ECC that is based on tandem activation of the RyRs by cytosolic and luminal Ca2+ through a ‘fire–diffuse–uptake–fire’ (or FDUF) mechanism: Ca2+ uptake by SR Ca2+ pumps at the propagation front elevates Ca2+ inside the SR locally, leading to luminal RyR sensitization and lowering of the cytosolic Ca2+ activation threshold.
Abstract
In atrial myocytes Ca2+ release during excitation–contraction coupling (ECC) is strikingly different from ventricular myocytes. In many species atrial myocytes lack a transverse tubule system, dividing the sarcoplasmic reticulum (SR) Ca2+ store into the peripheral subsarcolemmnal junctional (j‐SR) and the much more abundant central non‐junctional (nj‐SR) SR. Action potential (AP)‐induced Ca2+ entry activates Ca2+‐induced Ca2+ release (CICR) from j‐SR ryanodine receptor (RyR) Ca2+ release channels. Peripheral elevation of [Ca2+]i initiates CICR from nj‐SR and sustains propagation of CICR to the cell centre. Simultaneous confocal measurements of cytosolic ([Ca2+]i; with the fluorescent Ca2+ indicator rhod‐2) and intra‐SR ([Ca2+]SR; fluo‐5N) Ca2+ in rabbit atrial myocytes revealed that Ca2+ release from j‐SR resulted in a cytosolic Ca2+ transient of higher amplitude compared to release from nj‐SR; however, the degree of depletion of j‐SR [Ca2+]SR was smaller than nj‐SR [Ca2+]SR. Similarly, Ca2+ signals from individual release sites of the j‐SR showed a larger cytosolic amplitude (Ca2+ sparks) but smaller depletion (Ca2+ blinks) than release from nj‐SR. During AP‐induced Ca2+ release the rise of [Ca2+]i detected at individual release sites of the nj‐SR preceded the depletion of [Ca2+]SR, and during this latency period a transient elevation of [Ca2+]SR occurred. We propose that Ca2+ release from nj‐SR is activated by cytosolic and luminal Ca2+ (tandem RyR activation) via a novel ‘fire—diffuse–uptake–fire’ (FDUF) mechanism. This novel paradigm of atrial ECC predicts that Ca2+ uptake by sarco‐endoplasmic reticulum Ca2+‐ATPase (SERCA) at the propagation front elevates local [Ca2+]SR, leading to luminal RyR sensitization and lowering of the activation threshold for cytosolic CICR.

Key points
Smn+/− transgenic mouse is a model of the mildest form of spinal muscular atrophy.
Although there is a loss of spinal motoneurons in 11‐month‐old animals, muscular force is maintained.
This maintained muscular force is mediated by reinnervation of the denervated fibres by surviving motoneurons.
The spinal motoneurons in these animals do not show an increased susceptibility to death after nerve injury and they retain their regenerative capacity.
We conclude that the hypothesized immaturity of the neuromuscular system in this model cannot explain the loss of motoneurons by systematic die‐back.
Abstract
Spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and is the leading genetic cause of infantile death. Patients lack the SMN1 gene with the severity of the disease depending on the number of copies of the highly homologous SMN2 gene. Although motoneuron death in the Smn+/− transgenic mouse model of the mildest form of SMA, SMA type III, has been reported, we have used retrograde tracing of sciatic and femoral motoneurons in the hindlimb with recording of muscle and motor unit isometric forces to count the number of motoneurons with intact neuromuscular connections. Thereby, we investigated whether incomplete maturation of the neuromuscular system induced by survival motoneuron protein (SMN) defects is responsible for die‐back of axons relative to survival of motoneurons. First, a reduction of ∼30% of backlabelled motoneurons began relatively late, at 11 months of age, with a significant loss of 19% at 7 months. Motor axon die‐back was affirmed by motor unit number estimation. Loss of functional motor units was fully compensated by axonal sprouting to retain normal contractile force in four hindlimb muscles (three fast‐twitch and one slow‐twitch) innervated by branches of the sciatic nerve. Second, our evaluation of whether axotomy of motoneurons in the adult Smn+/− transgenic mouse increases their susceptibility to cell death demonstrated that all the motoneurons survived and they sustained their capacity to regenerate their nerve fibres. It is concluded the systematic die‐back of motoneurons that innervate both fast‐ and slow‐twitch muscle fibres is not related to immaturity of the neuromuscular system in SMA.

Key points
The functional importance of residues in loop G of the GABAA receptor has not been investigated. D43 and T47 in the α1 subunit are of particular significance as their structural modification inhibits activation by GABA.
While the T47C substitution had no significant effect, non‐conservative substitution of either residue (D43C or T47R) reduced the apparent potency of GABA.
Propofol potentiated maximal GABA‐evoked currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors. Non‐stationary variance analysis revealed a reduction in maximal GABA‐evoked Popen, suggesting impaired agonist efficacy.
Further analysis of α1(T47R)β2γ2 receptors revealed that the efficacy of the partial agonist THIP (4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridine‐3‐ol) relative to GABA was impaired.
GABA‐, THIP‐ and propofol‐evoked currents mediated by α1(T47R)β2γ2 receptors deactivated faster than those mediated by α1β2γ2 receptors, indicating that the mutation impairs agonist‐evoked gating.
Spontaneous gating caused by the β2(L285R) mutation was also reduced in α1(T47R)β2(L285R)γ2 compared to α1β2(L285R)γ2 receptors, confirming that α1(T47R) impairs gating independently of agonist activation.
Abstract
The modification of cysteine residues (substituted for D43 and T47) by 2‐aminoethyl methanethiosulfonate in the GABAA α1 subunit loop G is known to impair activation of α1β2γ2 receptors by GABA and propofol. While the T47C substitution had no significant effect, non‐conservative substitution of either residue (D43C or T47R) reduced the apparent potency of GABA. Propofol (1 μm), which potentiates sub‐maximal but not maximal GABA‐evoked currents mediated by α1β2γ2 receptors, also potentiated maximal currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors. Furthermore, the peak open probabilities of α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were reduced. The kinetics of macroscopic currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were characterised by slower desensitisation and faster deactivation. Similar changes in macroscopic current kinetics, together with a slower activation rate, were observed with the loop D α1(F64C) substitution, known to impair both efficacy and agonist binding, and when the partial agonist THIP (4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridine‐3‐ol) was used to activate WT or α1(T47R)β2γ2 receptors. Propofol‐evoked currents mediated by α1(T47R)β2γ2 and α1(F64C)β2γ2 receptors also exhibited faster deactivation than their WT counterparts, revealing that these substitutions impair gating through a mechanism independent of orthosteric binding. Spontaneous gating caused by the introduction of the β2(L285R) mutation was also reduced in α1(T47R)β2(L285R)γ2 compared to α1β2(L285R)γ2 receptors, confirming that α1(T47R) impairs gating independently of activation by any agonist. These findings implicate movement of the GABAA receptor α1 subunit's β1 strand during agonist‐dependent and spontaneous gating. Immobilisation of the β1 strand may provide a mechanism for the inhibition of gating by inverse agonists such as bicuculline.

Key points
Age significantly modifies the relationship between aortic pulse wave velocity and telomere length.
The differential relationships observed between aortic pulse wave velocity and telomere length in younger and older individuals suggest that the links between cellular and vascular ageing reflect a complex interaction between genetic and environmental factors acting over the life‐course.
Abstract
Ageing is associated with marked large artery stiffening. Telomere shortening, a marker of cellular ageing, is linked with arterial stiffening. However, the results of existing studies are inconsistent, possibly because of the confounding influence of variable exposure to cardiovascular risk factors. Therefore, we investigated the relationship between telomere length (TL) and aortic stiffness in well‐characterized, younger and older healthy adults, who were pre‐selected on the basis of having either low or high aortic pulse wave velocity (aPWV), a robust measure of aortic stiffness. Demographic, haemodynamic and biochemical data were drawn from participants in the Anglo‐Cardiff Collaborative Trial. Two age groups with an equal sex ratio were examined: those aged <30 years (younger) or >50 years (older). Separately for each age group and sex, DNA samples representing the highest (n = 125) and lowest (n = 125) extremes of aPWV (adjusted for blood pressure) were selected for analysis of leukocyte TL. Ultimately, this yielded complete phenotypic data on 904 individuals. In younger subjects, TL was significantly shorter in those with high aPWV vs. those with low aPWV (P = 0.017). By contrast, in older subjects, TL was significantly longer in those with high aPWV (P = 0.001). Age significantly modified the relationship between aPWV and TL (P

Key points
Heart rate is increased in chronic hypoxia and we tested whether this is the result of increased sympathetic nervous activity, reduced parasympathetic nervous activity, or a non‐autonomic mechanism.
In seven lowlanders, heart rate was measured at sea level and after 2 weeks at high altitude after individual and combined pharmacological inhibition of sympathetic and/or parasympathetic control of the heart.
Inhibition of parasympathetic control of the heart alone or in combination with inhibition of sympathetic control abolished the high altitude‐induced increase in heart rate.
Inhibition of sympathetic control of the heart alone did not prevent the high altitude‐induced increase in heart rate.
These results indicate that a reduced parasympathetic nervous activity is the main mechanism underlying the elevated heart rate in chronic hypoxia.
Abstract
Chronic hypoxia increases resting heart rate (HR), but the underlying mechanism remains incompletely understood. We investigated the relative contributions of the sympathetic and parasympathetic nervous systems, along with potential non‐autonomic mechanisms, by individual and combined pharmacological inhibition of muscarinic and/or β‐adrenergic receptors. In seven healthy lowlanders, resting HR was determined at sea level (SL) and after 15–18 days of exposure to 3454 m high altitude (HA) without drug intervention (control, CONT) as well as after intravenous administration of either propranolol (PROP), or glycopyrrolate (GLYC), or PROP and GLYC in combination (PROP+GLYC). Circulating noradrenaline concentration increased from 0.9 ± 0.4 nmol l−1 at SL to 2.7 ± 1.5 nmol l−1 at HA (P = 0.03). The effect of HA on HR depended on the type of autonomic inhibition (P = 0.006). Specifically, HR was increased at HA from 64 ± 10 to 74 ± 12 beats min−1 during the CONT treatment (P = 0.007) and from 52 ± 4 to 59 ± 5 beats min−1 during the PROP treatment (P

Key points
We report that the small molecule CK‐2066260 selectively slows the off‐rate of Ca2+ from fast skeletal muscle troponin, leading to increased myofibrillar Ca2+ sensitivity in fast skeletal muscle.
Rodents dosed with CK‐2066260 show increased hindlimb muscle force and power in response to submaximal rates of nerve stimulation in situ.
CK‐2066260 has no effect on free cytosolic [Ca2+] during contractions of isolated muscle fibres.
We conclude that fast skeletal muscle troponin sensitizers constitute a potential therapy to address an unmet need of improving muscle function in conditions of weakness and premature muscle fatigue.
Abstract
Skeletal muscle dysfunction occurs in many diseases and can lead to muscle weakness and premature muscle fatigue. Here we show that the fast skeletal troponin activator, CK‐2066260, counteracts muscle weakness by increasing troponin Ca2+ affinity, thereby increasing myofibrillar Ca2+ sensitivity. Exposure to CK‐2066260 resulted in a concentration‐dependent increase in the Ca2+ sensitivity of ATPase activity in isolated myofibrils and reconstituted hybrid sarcomeres containing fast skeletal muscle troponin C. Stopped‐flow experiments revealed a ∼2.7‐fold decrease in the Ca2+ off‐rate of isolated troponin complexes in the presence of CK‐2066260 (6 vs. 17 s−1 under control conditions). Isolated mouse flexor digitorum brevis fibres showed a rapidly developing, reversible and concentration‐dependent force increase at submaximal stimulation frequencies. This force increase was not accompanied by any changes in the free cytosolic [Ca2+] or its kinetics. CK‐2066260 induced a slowing of relaxation, which was markedly larger at 26°C than at 31°C and could be linked to the decreased Ca2+ off‐rate of troponin C. Rats dosed with CK‐2066260 showed increased hindlimb isometric and isokinetic force in response to submaximal rates of nerve stimulation in situ producing significantly higher absolute forces at low isokinetic velocities, whereas there was no difference in force at the highest velocities. Overall muscle power was increased and the findings are consistent with a lack of effect on crossbridge kinetics. In conclusion, CK‐2066260 acts as a fast skeletal troponin activator that may be used to increase muscle force and power in conditions of muscle weakness.

Key points
A mathematical model of a small conductance Ca2+‐activated potassium (SK) channel was developed and incorporated into a physiologically detailed ventricular myocyte model.
Ca2+‐sensitive K+ currents promote negative intracellular Ca2+ to membrane voltage (CAi2+→ Vm) coupling.
Increase of Ca2+‐sensitive K+ currents can be responsible for electromechanically discordant alternans and quasiperiodic oscillations at the cellular level.
At the tissue level, Turing‐type instability can occur when Ca2+‐sensitive K+ currents are increased.
Abstract
Cardiac alternans is a precursor to life‐threatening arrhythmias. Alternans can be caused by instability of the membrane voltage (Vm), instability of the intracellular Ca2+ ( Ca i2+) cycling, or both. Vm dynamics and Ca i2+ dynamics are coupled via Ca2+‐sensitive currents. In cardiac myocytes, there are several Ca2+‐sensitive potassium (K+) currents such as the slowly activating delayed rectifier current (IKs) and the small conductance Ca2+‐activated potassium (SK) current (ISK). However, the role of these currents in the development of arrhythmias is not well understood. In this study, we investigated how these currents affect voltage and Ca2+ alternans using a physiologically detailed computational model of the ventricular myocyte and mathematical analysis. We define the coupling between Vm and Ca i2+ cycling dynamics ( Ca i2+→Vm coupling) as positive (negative) when a larger Ca2+ transient at a given beat prolongs (shortens) the action potential duration (APD) of that beat. While positive coupling predominates at baseline, increasing IKs and ISK promote negative Ca i2+→Vm coupling at the cellular level. Specifically, when alternans is Ca2+‐driven, electromechanically (APD–Ca2+) concordant alternans becomes electromechanically discordant alternans as IKs or ISK increase. These cellular level dynamics lead to different types of spatially discordant alternans in tissue. These findings help to shed light on the underlying mechanisms of cardiac alternans especially when the relative strength of these currents becomes larger under pathological conditions or drug administrations.

Key points
To swallow food and liquid safely, airway protection is essential.
Upward and forward movements of the hyoid and larynx in the neck during swallowing vary in magnitude between individuals.
In healthy human adults, hyoid and laryngeal movements during swallowing were scaled by differences in initial upper airway area before swallowing.
Individuals increased laryngeal elevation during swallowing in response to increased airway opening before swallowing.
We show that when upper airway protection requirements change, individuals use an internal sensorimotor scaling system to adapt movements to maintain swallow safety.
Abstract
Hyoid and laryngeal movements contribute to laryngeal vestibule closure and upper oesophageal sphincter opening during swallowing. Evidence of an internal sensorimotor scaling system allowing individuals to achieve these functional goals is lacking. In speech, speakers adjust their articulatory movement magnitude according to the movement distance required to reach an articulatory target for intelligible speech. We investigated if swallowing is similar in that movement amplitude may be scaled by the functional goal for airway protection during swallowing, rather than by head and neck size. We hypothesized that healthy individuals adapt to their own anatomy by adjusting hyo‐laryngeal movements to achieve closure of the upper airway. We also investigated if individuals would automatically compensate for changes in their initial hyo‐laryngeal positions and area when head position was changed prior to swallowing. Videofluoroscopy was performed in 31 healthy adults. Using frame‐by‐frame motion analysis, anterior and superior hyoid and laryngeal displacement, and hyo‐laryngeal area were measured prior to and during swallowing. Kinematic measurements during swallowing were examined for relationships with pharyngeal neck length, and initial hyo‐laryngeal positions, length and area before swallowing. During swallowing, individuals altered laryngeal elevation magnitude to exceed hyoid elevation based on hyo‐laryngeal length before swallowing. Anterior laryngeal displacement was related to initial larynx distance from the spine, while hyoid elevation was predicted by pharyngeal neck length and initial hyoid distance from the mandible prior to the swallow. In conclusion, individuals automatically adapt hyo‐laryngeal movement during swallowing based on targets required for closing the hyo‐laryngeal area for safe swallowing.

Key points
The membrane‐associated guanylate kinase (MAGUK) family of synaptic scaffolding proteins anchor glutamate receptors at CNS synapses.
MAGUK removal via RNAi‐mediated knockdown in the CA1 hippocampal region in immature animals causes rapid and lasting reductions in glutamatergic transmission. In mature animals, the same manipulation has little acute effect.
The hippocampal dentate gyrus, a region with ongoing adult neurogenesis, is sensitive to MAGUK loss in mature animals, behaving like an immature CA1.
Over long time courses, removal of MAGUKs in CA1 causes reductions in glutamatergic transmission, indicating that synapses in mature animals require MAGUKs for anchoring glutamate receptors, but are much more stable.
These results demonstrate regional and developmental control of synapse stability and suggest the existence of a sensitive period of heightened hippocampal plasticity in CA1 of pre‐adolescent rodents, and in dentate gyrus throughout maturity.
Abstract
Fast excitatory transmission in the brain requires localization of glutamate receptors to synapses. The membrane‐associated guanylate kinase (MAGUK) family of synaptic scaffolding proteins is critical for localization of glutamate receptors to synapses. Although the MAGUKs are well‐studied in reduced preparations and young animals, few data exist on their role in adult animals. Here, we present a detailed developmental study of the role of the MAGUKs during rat development. We first confirmed by knockdown experiments that MAGUKs are essential for glutamatergic transmission in young animals and cultured slices, and an increase in postsynaptic density protein 95 (PSD‐95) by overexpression caused correlated increases in glutamatergic transmission. We found that CA1 synapses in adults, in contrast, were largely unaffected by overexpression of MAGUKs, and although adult CA1 synapses required MAGUKs to the same degree as synapses in young animals, this was only apparent over long time scales of knockdown. We additionally showed that overexpression of MAGUKs is likely to function to accelerate the developmental strengthening of excitatory transmission. Finally, we showed that adult dentate gyrus appears similar to immature CA1, demonstrating regional and developmental control of MAGUK dynamics. Together, these results demonstrate a period of juvenile instability at CA1 synapses, followed by a period of adult stability in which synapses are acutely unaffected by changes in MAGUK abundance.

Key points
Trans‐spinal polarization was recently introduced as a means to improve deficient spinal functions. However, only a few attempts have been made to examine the mechanisms underlying DC actions. We have now examined the effects of DC on two spinal modulatory systems, presynaptic inhibition and post‐activation depression, considering whether they might weaken exaggerated spinal reflexes and enhance excessively weakened ones.
Direct current effects were evoked by using local intraspinal DC application (0.3–0.4 μA) in deeply anaesthetized rats and were compared with the effects of trans‐spinal polarization (0.8–1.0 mA).
Effects of local intraspinal DC were found to be polarity dependent, as locally applied cathodal polarization enhanced presynaptic inhibition and post‐activation depression, whereas anodal polarization weakened them. In contrast, both cathodal and anodal trans‐spinal polarization facilitated them.
The results suggest some common DC‐sensitive mechanisms of presynaptic inhibition and post‐activation depression, because both were facilitated or depressed by DC in parallel.
Abstract
Direct current (DC) polarization has been demonstrated to alleviate the effects of various deficits in the operation of the central nervous system. However, the effects of trans‐spinal DC stimulation (tsDCS) have been investigated less extensively than the effects of transcranial DC stimulation, and their cellular mechanisms have not been elucidated. The main objectives of this study were, therefore, to extend our previous analysis of DC effects on the excitability of primary afferents and synaptic transmission by examining the effects of DC on two spinal modulatory feedback systems, presynaptic inhibition and post‐activation depression, in an anaesthetized rat preparation. Other objectives were to compare the effects of locally and trans‐spinally applied DC (locDC and tsDCS). Local polarization at the sites of terminal branching of afferent fibres was found to induce polarity‐dependent actions on presynaptic inhibition and post‐activation depression, as cathodal locDC enhanced them and anodal locDC depressed them. In contrast, tsDCS modulated presynaptic inhibition and post‐activation depression in a polarity‐independent fashion because both cathodal and anodal tsDCS facilitated them. The results show that the local presynaptic actions of DC might counteract both excessively strong and excessively weak monosynaptic actions of group Ia and cutaneous afferents. However, they indicate that trans‐spinally applied DC might counteract the exaggerated spinal reflexes but have an adverse effect on pathologically weakened spinal activity by additional presynaptic weakening. The results are also relevant for the analysis of the basic properties of presynaptic inhibition and post‐activation depression because they indicate that some common DC‐sensitive mechanisms contribute to them.

Key points
The cerebellum is the core structure controlling gaze stability. Chronic cerebellar diseases and acute alcohol intoxication affect cerebellar function, inducing, among others, gaze instability as gaze‐evoked nystagmus.
Gaze‐evoked nystagmus is characterized by increased centripetal eye‐drift. It is used as an important diagnostic sign for patients with cerebellar degeneration and to assess the ‘driving while intoxicated’ condition.
We quantified the effect of alcohol on gaze‐holding using an approach allowing, for the first time, the comparison of deficits induced by alcohol intoxication and cerebellar degeneration.
Our results showed that alcohol intoxication induces a two‐fold increase of centripetal eye‐drift.
We establish analysis techniques for using controlled alcohol intake as a model to support the study of cerebellar deficits.
The observed similarity between the effect of alcohol and the clinical signs observed in cerebellar patients suggests a possible pathomechanism for gaze‐holding deficits.
Abstract
Gaze‐evoked nystagmus (GEN) is an ocular‐motor finding commonly observed in cerebellar disease, characterized by increased centripetal eye‐drift with centrifugal correcting saccades at eccentric gaze. With cerebellar degeneration being a rare and clinically heterogeneous disease, data from patients are limited. We hypothesized that a transient inhibition of cerebellar function by defined amounts of alcohol may provide a suitable model to study gaze‐holding deficits in cerebellar disease. We recorded gaze‐holding at varying horizontal eye positions in 15 healthy participants before and 30 min after alcohol intake required to reach 0.6‰ blood alcohol content (BAC). Changes in ocular‐motor behaviour were quantified measuring eye‐drift velocity as a continuous function of gaze eccentricity over a large range (±40 deg) of horizontal gaze angles and characterized using a two‐parameter tangent model. The effect of alcohol on gaze stability was assessed analysing: (1) overall effects on the gaze‐holding system, (2) specific effects on each eye and (3) differences between gaze angles in the temporal and nasal hemifields. For all subjects, alcohol consumption induced gaze instability, causing a two‐fold increase [2.21 (0.55), median (median absolute deviation); P = 0.002] of eye‐drift velocity at all eccentricities. Results were confirmed analysing each eye and hemifield independently. The alcohol‐induced transient global deficit in gaze‐holding matched the pattern previously described in patients with late‐onset cerebellar degeneration. Controlled intake of alcohol seems a suitable disease model to study cerebellar GEN. With alcohol resulting in global cerebellar hypofunction, we hypothesize that patients matching the gaze‐holding behaviour observed here suffered from diffuse deficits in the gaze‐holding system as well.

Key points
In human sperm, proton flux across the membrane is controlled by the voltage‐gated proton channel Hv1.
We show that sperm harbour both Hv1 and an N‐terminally cleaved isoform termed Hv1Sper.
The pH‐control of Hv1Sper and Hv1 is distinctively different.
Hv1Sper and Hv1 can form heterodimers that combine features of both constituents.
Cleavage and heterodimerization of Hv1 might represent an adaptation to the specific requirements of pH control in sperm.
Abstract
In human sperm, the voltage‐gated proton channel Hv1 controls the flux of protons across the flagellar membrane. Here, we show that sperm harbour Hv1 and a shorter isoform, termed Hv1Sper. Hv1Sper is generated from Hv1 by removal of 68 amino acids from the N‐terminus by post‐translational proteolytic cleavage. The pH‐dependent gating of the channel isoforms is distinctly different. In both Hv1 and Hv1Sper, the conductance–voltage relationship is determined by the pH difference across the membrane (∆pH). However, simultaneous changes in intracellular and extracellular pH that leave ΔpH constant strongly shift the activation curve of Hv1Sper but not that of Hv1, demonstrating that cleavage of the N‐terminus tunes pH sensing in Hv1. Moreover, we show that Hv1 and Hv1Sper assemble as heterodimers that combine features of both constituents. We suggest that cleavage and heterodimerization of Hv1 represents an adaptation to the specific requirements of pH control in sperm.

Key points
The nucleus incertus is a key node of the brainstem circuitry involved in hippocampal theta rhythmicity.
Synchronisation exists between the nucleus incertus and hippocampal activities during theta periods.
By the Granger causality analysis, we demonstrated a directional information flow between theta rhythmical neurons in the nucleus incertus and the hippocampus in theta‐on states.
The electrical stimulation of the nucleus incertus is also able to evoke a phase reset of the hippocampal theta wave.
Our data suggest that the nucleus incertus is a key node of theta generation and the modulation network.
Abstract
In recent years, a body of evidence has shown that the nucleus incertus (NI), in the dorsal tegmental pons, is a key node of the brainstem circuitry involved in hippocampal theta rhythmicity. Ascending reticular brainstem system activation evokes hippocampal theta rhythm with coupled neuronal activity in the NI. In a recent paper, we showed three populations of neurons in the NI with differential firing during hippocampal theta activation. The objective of this work was to better evaluate the causal relationship between the activity of NI neurons and the hippocampus during theta activation in order to further understand the role of the NI in the theta network. A Granger causality analysis was run to determine whether hippocampal theta activity with sensory‐evoked theta depends on the neuronal activity of the NI, or vice versa. The analysis showed causal interdependence between the NI and the hippocampus during theta activity, whose directional flow depended on the different neuronal assemblies of the NI. Whereas type I and II NI neurons mainly acted as receptors of hippocampal information, type III neuronal activity was the predominant source of flow between the NI and the hippocampus in theta states. We further determined that the electrical activation of the NI was able to reset hippocampal waves with enhanced theta‐band power, depending on the septal area. Collectively, these data suggest that hippocampal theta oscillations after sensory activation show dependence on NI neuron activity, which could play a key role in establishing optimal conditions for memory encoding.

Abstract
This is the second of the two White Papers from the fourth UC Davis Cardiovascular Symposium Systems Approach to Understanding Cardiac Excitation–Contraction Coupling and Arrhythmias (3–4 March 2016), a biennial event that brings together leading experts in different fields of cardiovascular research. The theme of the 2016 symposium was ‘K+ channels and regulation’, and the objectives of the conference were severalfold: (1) to identify current knowledge gaps; (2) to understand what may go wrong in the diseased heart and why; (3) to identify possible novel therapeutic targets; and (4) to further the development of systems biology approaches to decipher the molecular mechanisms and treatment of cardiac arrhythmias. The sessions of the Symposium focusing on the functional roles of the cardiac K+ channel in health and disease, as well as K+ channels as therapeutic targets, were contributed by Ye Chen‐Izu, Gideon Koren, James Weiss, David Paterson, David Christini, Dobromir Dobrev, Jordi Heijman, Thomas O'Hara, Crystal Ripplinger, Zhilin Qu, Jamie Vandenberg, Colleen Clancy, Isabelle Deschenes, Leighton Izu, Tamas Banyasz, Andras Varro, Heike Wulff, Eleonora Grandi, Michael Sanguinetti, Donald Bers, Jeanne Nerbonne and Nipavan Chiamvimonvat as speakers and panel discussants. This article summarizes state‐of‐the‐art knowledge and controversies on the functional roles of cardiac K+ channels in normal and diseased heart. We endeavour to integrate current knowledge at multiple scales, from the single cell to the whole organ levels, and from both experimental and computational studies.

Key points
Despite an attenuated fluctuation in ovarian hormone concentrations in well‐trained women, one in two of such women believe their menstrual cycle negatively impacts training and performance.
Forthcoming large international events will expose female athletes to hot environments, and studies evaluating aerobic exercise performance in such environments across the menstrual cycle are sparse, with mixed findings.
We have identified that autonomic heat loss responses at rest and during fixed‐intensity exercise in well‐trained women are not affected by menstrual cycle phase, but differ between dry and humid heat.
Furthermore, exercise performance is not different across the menstrual cycle, yet is lower in humid heat, in conjunction with reduced evaporative cooling.
Menstrual cycle phase does not appear to affect exercise performance in the heat in well‐trained women, but humidity impairs performance, probably due to reduced evaporative power.
Abstract
We studied thermoregulatory responses of ten well‐trained [V̇O2 max , 57 (7) ml min−1 kg−1] eumenorrheic women exercising in dry and humid heat, across their menstrual cycle. They completed four trials, each of resting and cycling at fixed intensities (125 and 150 W), to assess autonomic regulation, then self‐paced intensity (30 min work trial), to assess behavioural regulation. Trials were in early‐follicular (EF) and mid‐luteal (ML) phases in dry (DRY) and humid (HUM) heat matched for wet bulb globe temperature (WBGT, 27°C). During rest and fixed‐intensity exercise, rectal temperature was ∼0.2°C higher in ML than EF (P

Key points
The calcium‐activated chloride channel TMEM16A provides a pathway for chloride ion movements that are key in preventing polyspermy, allowing fluid secretion, controlling blood pressure, and enabling gastrointestinal activity.
TMEM16A is opened by voltage‐dependent calcium binding and regulated by permeant anions and intracellular protons.
Here we show that a low proton concentration reduces TMEM16A activity while maximum activation is obtained when the external proton concentration is high.
In addition, protonation conditions determine the open probability of TMEM16A without changing its calcium sensitivity. External glutamic acid 623 (E623) is key for TMEM16A's ability to respond to external protons.
At physiological pH, E623 is un‐protonated and TMEM16A is activated when intracellular calcium increases; however, under acidic conditions E623 is partially protonated and works synergistically with intracellular calcium to activate the channel. These findings are critical for understanding physiological and pathological processes that involve changes in pH and chloride flux via TMEM16A.
Abstract
Transmembrane protein 16A (TMEM16A), also known as ANO1, the pore‐forming subunit of a Ca2+‐dependent Cl− channel (CaCC), is activated by direct, voltage‐dependent, binding of intracellular Ca2+. Endogenous CaCCs are regulated by extracellular protons; however, the molecular basis of such regulation remains unidentified. Here, we evaluated the effects of different extracellular proton concentrations ([H+]o) on mouse TMEM16A expressed in HEK‐293 cells using whole‐cell and inside‐out patch‐clamp recordings. We found that increasing the [H+]o from 10−10 to 10−5.5 m caused a progressive increase in the chloride current (ICl) that is described by titration of a protonatable site with pK = 7.3. Protons regulate TMEM16A in a voltage‐independent manner, regardless of channel state (open or closed), and without altering its apparent Ca2+ sensitivity. Noise analysis showed that protons regulate TMEM16A by tuning its open probability without modifying the single channel current. We found a robust reduction of the proton effect at high [Ca2+]i. To identify protonation targets we mutated all extracellular glutamate and histidine residues and 4 of 11 aspartates. Most mutants were sensitive to protons. However, mutation that substituted glutamic acid (E) for glutamine (Q) at amino acid position 623 (E623Q) displayed a titration curve shifted to the left relative to wild type channels and the ICl was nearly insensitive to proton concentrations between 10−5.5 and 10−9.0 m. Additionally, ICl of the mutant containing an aspartic acid (D) to asparagine (N) substitution at position 405 (D405N) mutant was partially inhibited by a proton concentration of 10−5.5 m, but 10−9.0 m produced the same effect as in wild type. Based on our findings we propose that external protons titrate glutamic acid 623, which enables voltage activation of TMEM16A at non‐saturating [Ca2+]i.

Key points
The cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF), forms a gated pathway for chloride movement regulated by intracellular ATP.
To understand better CFTR function, we investigated the regulation of channel openings by intracellular pH.
We found that short‐lived channel closures during channel openings represent subtle changes in the structure of CFTR that are regulated by intracellular pH, in part, at ATP‐binding site 1 formed by the nucleotide‐binding domains.
Our results provide a framework for future studies to understand better the regulation of channel openings, the dysfunction of CFTR in CF and the action of drugs that repair CFTR gating defects.
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP‐gated Cl− channel defective in the genetic disease cystic fibrosis (CF). The gating behaviour of CFTR is characterized by bursts of channel openings interrupted by brief, flickery closures, separated by long closures between bursts. Entry to and exit from an open burst is controlled by the interaction of ATP with two ATP‐binding sites, sites 1 and 2, in CFTR. To understand better the kinetic basis of CFTR intraburst gating, we investigated the single‐channel activity of human CFTR at different intracellular pH (pHi) values. When compared with the control (pHi 7.3), acidifying pHi to 6.3 or alkalinizing pHi to 8.3 and 8.8 caused small reductions in the open‐time constant (τo) of wild‐type CFTR. By contrast, the fast closed‐time constant (τcf), which describes the short‐lived closures that interrupt open bursts, was greatly increased at pHi 5.8 and 6.3. To analyse intraburst kinetics, we used linear three‐state gating schemes. All data were satisfactorily modelled by the C1 ↔ O ↔ C2 kinetic scheme. Changing the intracellular ATP concentration was without effect on τo, τcf and their responses to pHi changes. However, mutations that disrupt the interaction of ATP with ATP‐binding site 1, including K464A, D572N and the CF‐associated mutation G1349D all abolished the prolongation of τcf at pHi 6.3. Taken together, our data suggest that the regulation of CFTR intraburst gating is distinct from the ATP‐dependent mechanism that controls channel opening and closing. However, our data also suggest that ATP‐binding site 1 modulates intraburst gating.

Key points
Our objective was to quantify endothelial function (via brachial artery flow‐mediated dilatation) at sea level (344 m) and high altitude (3800 m) at rest and following both maximal exercise and 30 min of moderate‐intensity cycling exercise with and without administration of an α1‐adrenergic blockade.
Brachial endothelial function did not differ between sea level and high altitude at rest, nor following maximal exercise.
At sea level, endothelial function decreased following 30 min of moderate‐intensity exercise, and this decrease was abolished with α1‐adrenergic blockade. At high altitude, endothelial function did not decrease immediately after 30 min of moderate‐intensity exercise, and administration of α1‐adrenergic blockade resulted in an increase in flow‐mediated dilatation.
Our data indicate that post‐exercise endothelial function is modified at high altitude (i.e. prolonged hypoxaemia). The current study helps to elucidate the physiological mechanisms associated with high‐altitude acclimatization, and provides insight into the relationship between sympathetic nervous activity and vascular endothelial function.
Abstract
We examined the hypotheses that (1) at rest, endothelial function would be impaired at high altitude compared to sea level, (2) endothelial function would be reduced to a greater extent at sea level compared to high altitude after maximal exercise, and (3) reductions in endothelial function following moderate‐intensity exercise at both sea level and high altitude are mediated via an α1‐adrenergic pathway. In a double‐blinded, counterbalanced, randomized and placebo‐controlled design, nine healthy participants performed a maximal‐exercise test, and two 30 min sessions of semi‐recumbent cycling exercise at 50% peak output following either placebo or α1‐adrenergic blockade (prazosin; 0.05 mg kg −1). These experiments were completed at both sea‐level (344 m) and high altitude (3800 m). Blood pressure (finger photoplethysmography), heart rate (electrocardiogram), oxygen saturation (pulse oximetry), and brachial artery blood flow and shear rate (ultrasound) were recorded before, during and following exercise. Endothelial function assessed by brachial artery flow‐mediated dilatation (FMD) was measured before, immediately following and 60 min after exercise. Our findings were: (1) at rest, FMD remained unchanged between sea level and high altitude (placebo P = 0.287; prazosin: P = 0.110); (2) FMD remained unchanged after maximal exercise at sea level and high altitude (P = 0.244); and (3) the 2.9 ± 0.8% (P = 0.043) reduction in FMD immediately after moderate‐intensity exercise at sea level was abolished via α1‐adrenergic blockade. Conversely, at high altitude, FMD was unaltered following moderate‐intensity exercise, and administration of α1‐adrenergic blockade elevated FMD (P = 0.032). Our results suggest endothelial function is differentially affected by exercise when exposed to hypobaric hypoxia. These findings have implications for understanding the chronic impacts of hypoxaemia on exercise, and the interactions between the α1‐adrenergic pathway and endothelial function.

Key points
Arrhythmias result from disruptions to cardiac electrical activity, although the factors that control cellular action potentials are incompletely understood.
We combined mathematical modelling with experiments in heart cells from guinea pigs to determine how cellular electrical activity is regulated.
A mismatch between modelling predictions and the experimental results allowed us to construct an improved, more predictive mathematical model.
The balance between two particular potassium currents dictates how heart cells respond to perturbations and their susceptibility to arrhythmias.
Abstract
Imbalances of ionic currents can destabilize the cardiac action potential and potentially trigger lethal cardiac arrhythmias. In the present study, we combined mathematical modelling with information‐rich dynamic clamp experiments to determine the regulation of action potential morphology in guinea pig ventricular myocytes. Parameter sensitivity analysis was used to predict how changes in ionic currents alter action potential duration, and these were tested experimentally using dynamic clamp, a technique that allows for multiple perturbations to be tested in each cell. Surprisingly, we found that a leading mathematical model, developed with traditional approaches, systematically underestimated experimental responses to dynamic clamp perturbations. We then re‐parameterized the model using a genetic algorithm, which allowed us to estimate ionic current levels in each of the cells studied. This unbiased model adjustment consistently predicted an increase in the rapid delayed rectifier K+ current and a drastic decrease in the slow delayed rectifier K+ current, and this prediction was validated experimentally. Subsequent simulations with the adjusted model generated the clinically relevant prediction that the slow delayed rectifier is better able to stabilize the action potential and suppress pro‐arrhythmic events than the rapid delayed rectifier. In summary, iterative coupling of simulations and experiments enabled novel insight into how the balance between cardiac K+ currents influences ventricular arrhythmia susceptibility.

Key points
Following the genetically controlled formation of neuronal circuits, early firing activity guides the development of sensory maps in the auditory, visual and somatosensory system. However, it is not clear whether the activity of central auditory neurons is specifically regulated depending on the position within the sensory map.
In the ventral cochlear nucleus, the first central station along the auditory pathway, we describe a mechanism through which paracrine ATP signalling enhances firing in a cell‐specific and tonotopically‐determined manner.
Developmental down‐regulation of P2X2/3R currents along the tonotopic axis occurs simultaneously with an increase in AMPA receptor currents, suggesting a high‐to‐low frequency maturation pattern.
Facilitated action potential (AP) generation, measured as higher firing rate, shorter EPSP‐AP delay in vivo and shorter AP latency in slice experiments, is consistent with increased synaptic efficacy caused by ATP.
The long lasting change in intrinsic neuronal excitability is mediated by the heteromeric P2X2/3 receptors.
Abstract
Synaptic refinement and strengthening are activity‐dependent processes that establish orderly arranged cochleotopic maps throughout the central auditory system. The maturation of auditory brainstem circuits is guided by action potentials (APs) arising from the inner hair cells in the developing cochlea. The AP firing of developing central auditory neurons can be modulated by paracrine ATP signalling, as shown for the cochlear nucleus bushy cells and principal neurons in the medial nucleus of the trapezoid body. However, it is not clear whether neuronal activity may be specifically regulated with respect to the nuclear tonotopic position (i.e. sound frequency selectivity). Using slice recordings before hearing onset and in vivo recordings with iontophoretic drug applications after hearing onset, we show that cell‐specific purinergic modulation follows a precise tonotopic pattern in the ventral cochlear nucleus of developing gerbils. In high‐frequency regions, ATP responsiveness diminished before hearing onset. In low‐to‐mid frequency regions, ATP modulation persisted after hearing onset in a subset of low‐frequency bushy cells (characteristic frequency

Key points
Endothelial inwardly rectifying K+ (Kir2.1) channels regulate flow‐induced vasodilatation via nitric oxide (NO) in mouse mesenteric resistance arteries.
Deficiency of Kir2.1 channels results in elevated blood pressure and increased vascular resistance.
Flow‐induced vasodilatation in human resistance arteries is also regulated by inwardly rectifying K+ channels.
This study presents the first direct evidence that Kir channels play a critical role in physiological endothelial responses to flow.
Abstract
Inwardly rectifying K+ (Kir) channels are known to be sensitive to flow, but their role in flow‐induced endothelial responses is not known. The goal of this study is to establish the role of Kir channels in flow‐induced vasodilatation and to provide first insights into the mechanisms responsible for Kir signalling in this process. First, we establish that primary endothelial cells isolated from murine mesenteric arteries express functional Kir2.1 channels sensitive to shear stress. Then, using the Kir2.1+/− heterozygous mouse model, we establish that downregulation of Kir2.1 results in significant decrease in shear‐activated Kir currents and inhibition of endothelium‐dependent flow‐induced vasodilatation (FIV) assayed in pressurized mesenteric arteries pre‐constricted with endothelin‐1. Deficiency in Kir2.1 also results in the loss of flow‐induced phosphorylation of eNOS and Akt, as well as inhibition of NO generation. All the effects are fully rescued by endothelial cell (EC)‐specific overexpression of Kir2.1. A component of FIV that is Kir independent is abrogated by blocking Ca2+‐sensitive K+ channels. Kir2.1 has no effect on endothelium‐independent and K+‐induced vasodilatation in denuded arteries. Kir2.1+/− mice also show increased mean blood pressure measured by carotid artery cannulation and increased microvascular resistance measured using a tail‐cuff. Importantly, blocking Kir channels also inhibits flow‐induced vasodilatation in human subcutaneous adipose microvessels. Endothelial Kir channels contribute to FIV of mouse mesenteric arteries via an NO‐dependent mechanism, whereas Ca2+‐sensitive K+ channels mediate FIV via an NO‐independent pathway. Kir2 channels also regulate vascular resistance and blood pressure. Finally, Kir channels also contribute to FIV in human subcutaneous microvessels.

Key points
Rodent models of intrauterine growth restriction (IUGR) successfully identify mechanisms that can lead to short‐term and long‐term detrimental cardiomyopathies but differences between rodent and human cardiac physiology and placental‐fetal development indicate a need for models in precocial species for translation to human development.
We developed a baboon model for IUGR studies using a moderate 30% global calorie restriction of pregnant mothers and used cardiac magnetic resonance imaging to evaluate offspring heart function in early adulthood.
Impaired diastolic and systolic cardiac function was observed in IUGR offspring with differences between male and female subjects, compared to their respective controls.
Aspects of cardiac impairment found in the IUGR offspring were similar to those found in normal controls in a geriatric cohort.
Understanding early cardiac biomarkers of IUGR using non‐invasive imaging in this susceptible population, especially taking into account sexual dimorphisms, will aid recognition of the clinical presentation, development of biomarkers suitable for use in humans and management of treatment strategies.
Abstract
Extensive rodent studies have shown that reduced perinatal nutrition programmes chronic cardiovascular disease. To enable translation to humans, we developed baboon offspring cohorts from mothers fed ad libitum (control) or 70% of the control ad libitum diet in pregnancy and lactation, which were growth restricted at birth. We hypothesized that intrauterine growth restriction (IUGR) offspring hearts would show impaired function and a premature ageing phenotype. We studied IUGR baboons (8 male, 8 female, 5.7 years), control offspring (8 male, 8 female, 5.6 years – human equivalent approximately 25 years), and normal elderly (OLD) baboons (6 male, 6 female, mean 15.9 years). Left ventricular (LV) morphology and systolic and diastolic function were evaluated with cardiac MRI and normalized to body surface area. Two‐way ANOVA by group and sex (with P

Key points
In the adult turtle spinal cord, action potential generation in motoneurones is inhibited by spillover of serotonin to extrasynaptic serotonin 1A (5‐HT1A) receptors at the axon initial segment. We explored whether ingestion of the 5‐HT1A receptor partial agonist, buspirone, decreases motoneurone excitability in humans.
Following ingestion of buspirone, two tests of motoneurone excitability showed decreases. F‐wave areas and persistence in an intrinsic muscle of the hand were reduced, as was the area of cervicomedullary motor evoked potentials in biceps brachii.
Our findings suggest that activation of 5‐HT1A receptors depresses human motoneurone excitability. Such a depression could contribute to decreased motoneurone output during fatiguing exercise if there is high serotonergic drive to the motoneurones.
Abstract
Intense serotonergic drive in the turtle spinal cord results in serotonin spillover to the axon initial segment of the motoneurones where it activates serotonin 1A (5‐HT1A) receptors and inhibits generation of action potentials. We examined whether activation of 5‐HT1A receptors decreases motoneurone excitability in humans by determining the effects of a 5‐HT1A receptor partial agonist, buspirone, on F waves and cervicomedullary motor evoked potentials (CMEPs). In a placebo‐controlled double‐blind study, 10 participants were tested on two occasions where either placebo or 20 mg of buspirone was administered orally. The ulnar nerve was stimulated supramaximally to evoke F waves in abductor digiti minimi (ADM). CMEPs and the maximal M wave were elicited in biceps brachii by cervicomedullary stimulation and brachial plexus stimulation, respectively. Following buspirone intake, F‐wave area and persistence, as well as CMEP area, were significantly decreased. The mean post‐pill difference in normalized F‐wave areas and persistence between buspirone and placebo days was –27% (–42, –12; 95% confidence interval) and –9% (–16, –2), respectively. The mean post‐pill difference in normalized CMEP area between buspirone and placebo days showed greater variation and was –31% (–60, –2). In conclusion, buspirone reduces motoneurone excitability in humans probably via activation of 5‐HT1A receptors at the axon initial segment. This has implications for motor output during high drive to the motoneurones when serotonin may spill over to these inhibitory receptors and consequently inhibit motoneurone output. Such a mechanism could potentially contribute to fatigue with exercise.

Key points
Incomplete development of the neural circuits that control breathing contributes to respiratory disorders in pre‐term infants. Manifestations include respiratory instability, prolonged apnoeas and poor ventilatory responses to stimuli.
Based on evidence suggesting that omega‐3 polyunsaturated fatty acids (n‐3 PUFA) improves brain development, we determined whether n‐3 PUFA supplementation (via the maternal diet) improves respiratory function in 10–11‐day‐old rat pups.
n‐3 PUFA treatment prolonged apnoea duration but augmented the relative pulmonary surface area and the ventilatory response to hypoxia. During hypoxia, the drop in body temperature measured in treated pups was 1 °C less than in controls. n‐3 PUFA treatment also reduced microglia cell density in the brainstem.
Although heterogeneous, the results obtained in rat pups constitute a proof of concept that n‐3 PUFA supplementation can have positive effects on neonatal respiration. This includes a more sustained hypoxic ventilatory response and a decreased respiratory inhibition during laryngeal chemoreflex.
Abstract
Most pre‐term infants present respiratory instabilities and apnoeas as a result of incomplete development of the neural circuits that control breathing. Because omega‐3 polyunsaturated fatty acids (n‐3 PUFA) benefit brain development, we hypothesized that n‐3 PUFA supplementation (via the maternal diet) improves respiratory function in rat pups. Pups received n‐3 PUFA supplementation from an enriched diet (13 g kg−1 of n‐3 PUFA) administered to the mother from birth until the experiments were performed (postnatal days 10–11). Controls received a standard diet (0.3 g kg−1 of n‐3 PUFA). Breathing was measured in intact pups at rest and during hypoxia (FiO2 = 0.12; 20 min) using whole body plethysmography. The duration of apnoeas induced by stimulating the laryngeal chemoreflex (LCR) was measured under anaesthesia. Lung morphology was compared between groups. Maternal n‐3 PUFA supplementation effectively raised n‐3 PUFA levels above control levels both in the blood and brainstem of pups. In intact, resting pups, n‐3 PUFA increased the frequency and duration of apnoeas, especially in females. During hypoxia, n‐3 PUFA supplemented pups hyperventilated 23% more than controls; their anapyrexic response was 1 °C less than controls. In anaesthetized pups, n‐3 PUFA shortened the duration of LCR‐induced apnoeas by 32%. The relative pulmonary surface area of n‐3 PUFA supplemented pups was 12% higher than controls. Although n‐3 PUFA supplementation augments apnoeas, there is no clear evidence of deleterious consequences on these pups. Based on the improved lung architecture and responses to respiratory challenges, this neonatal treatment appears to be beneficial to the offspring. However, further experiments are necessary to establish its overall safety.

Key points
Fibrosis occurs secondary to many skeletal muscle diseases and injuries, and can alter muscle function.
It is unknown how collagen, the most abundant extracellular structural protein, alters its organization during fibrosis.
Quantitative and qualitative high‐magnification electron microscopy shows that collagen is organized into perimysial cables which increase in number in a model of fibrosis, and cables have unique interactions with collagen‐producing cells.
Fibrotic muscles are stiffer and have a higher concentration of collagen‐producing cells.
These results improve our understanding of the organization of fibrotic skeletal muscle extracellular matrix and identify novel structures that might be targeted by antifibrotic therapy.
Abstract
Skeletal muscle extracellular matrix (ECM) structure and organization are not well understood, yet the ECM plays an important role in normal tissue homeostasis and disease processes. Fibrosis is common to many muscle diseases and is typically quantified based on an increase in ECM collagen. Through the use of multiple imaging modalities and quantitative stereology, we describe the structure and composition of wild‐type and fibrotic ECM, we show that collagen in the ECM is organized into large bundles of fibrils, or collagen cables, and the number of these cables (but not their size) increases in desmin knockout muscle (a fibrosis model). The increase in cable number is accompanied by increased muscle stiffness and an increase in the number of collagen producing cells. Unique interactions between ECM cells and collagen cables were also observed and reconstructed by serial block face scanning electron microscopy. These results demonstrate that the muscle ECM is more highly organized than previously reported. Therapeutic strategies for skeletal muscle fibrosis should consider the organization of the ECM to target the structures and cells contributing to fibrotic muscle function.

Key points
Cardiac vagal tone is a strong predictor of health, although its central origins are unknown.
Respiratory‐linked fluctuations in cardiac vagal tone give rise to respiratory sinus arryhthmia (RSA), with maximum tone in the post‐inspiratory phase of respiration.
In the present study, we investigated whether respiratory modulation of cardiac vagal tone is intrinsically linked to post‐inspiratory respiratory control using the unanaesthetized working heart‐brainstem preparation of the rat.
Abolition of post‐inspiration, achieved by inhibition of the pontine Kolliker‐Fuse nucleus, removed post‐inspiratory peaks in efferent cardiac vagal activity and suppressed RSA, whereas substantial cardiac vagal tone persisted. After transection of the caudal pons, part of the remaining tone was removed by inhibition of nucleus of the solitary tract.
We conclude that cardiac vagal tone depends upon at least 3 sites of the pontomedullary brainstem and that a significant proportion arises independently of RSA.
Abstract
Cardiac vagal tone is a strong predictor of health, although its central origins are unknown. The rat working heart‐brainstem preparation shows strong cardiac vagal tone and pronounced respiratory sinus arrhythmia. In this preparation, recordings from the cut left cardiac vagal branch showed efferent activity that peaked in post‐inspiration, ∼0.5 s before the cyclic minimum in heart rate (HR). We hypothesized that respiratory modulation of cardiac vagal tone and HR is intrinsically linked to the generation of post‐inspiration. Neurons in the pontine Kölliker‐Fuse nucleus (KF) were inhibited with bilateral microinjections of isoguvacine (50–70 nl, 10 mm) to remove the post‐inspiratory phase of respiration. This also abolished the post‐inspiratory peak of cardiac vagal discharge (and cyclical HR modulation), although a substantial level of activity remained. In separate preparations with intact cardiac vagal branches but sympathetically denervated by thoracic spinal pithing, cardiac chronotropic vagal tone was quantified by HR compared to its final level after systemic atropine (0.5 μm). Bilateral KF inhibition removed 88% of the cyclical fluctuation in HR but, on average, only 52% of the chronotropic vagal tone. Substantial chronotropic vagal tone also remained after transection of the brainstem through the caudal pons. Subsequent bilateral isoguvacine injections into the nucleus of the solitary tract further reduced vagal tone: remaining sources were untraced. We conclude that cardiac vagal tone depends on neurons in at least three sites of the pontomedullary brainstem, and much of it arises independently of respiratory sinus arrhythmia.

Key points
The endothelium plays a pivotal role in the vascular response to chemical and mechanical stimuli.
The endothelium is exquisitely sensitive to ACh, although the physiological significance of ACh‐induced activation of the endothelium is unknown.
In the present study, we investigated the mechanisms of flow‐mediated endothelial calcium signalling.
Our data establish that flow‐mediated endothelial calcium responses arise from the autocrine action of non‐neuronal ACh released by the endothelium.
Abstract
Circulating blood generates frictional forces (shear stress) on the walls of blood vessels. These frictional forces critically regulate vascular function. The endothelium senses these frictional forces and, in response, releases various vasodilators that relax smooth muscle cells in a process termed flow‐mediated dilatation. Although some elements of the signalling mechanisms have been identified, precisely how flow is sensed and transduced to cause the release of relaxing factors is poorly understood. By imaging signalling in large areas of the endothelium of intact arteries, we show that the endothelium responds to flow by releasing ACh. Once liberated, ACh acts to trigger calcium release from the internal store in endothelial cells, nitric oxide production and artery relaxation. Flow‐activated release of ACh from the endothelium is non‐vesicular and occurs via organic cation transporters. ACh is generated following mitochondrial production of acetylCoA. Thus, we show ACh is an autocrine signalling molecule released from endothelial cells, and identify a new role for the classical neurotransmitter in endothelial mechanotransduction.

Key points
β‐Adrenergic receptor agonists such as isoproterenol induce cutaneous vasodilatation and sweating in humans, but the mechanisms underpinning this response remain unresolved.
Using intradermal microdialysis, we evaluated the roles of nitric oxide synthase (NOS) and cyclooxygenase (COX) in β‐adrenergic cutaneous vasodilatation and sweating elicited by administration of isoproterenol.
We show that while NOS contributes to β‐adrenergic cutaneous vasodilatation, COX restricts cutaneous vasodilatation.
We also show that combined inhibition of NOS and COX augments β‐adrenergic sweating
These new findings advance our basic knowledge regarding the physiological control of cutaneous blood flow and sweating, and provide important and new information to better understand the physiological significance of β‐adrenergic receptors in the skin.
Abstract
β‐Adrenergic receptor agonists such as isoproterenol can induce cutaneous vasodilatation and sweating in humans, but the mechanisms underpinning this response remain unresolved. We evaluated the hypotheses that (1) nitric oxide synthase (NOS) contributes to β‐adrenergic cutaneous vasodilatation, whereas cyclooxygenase (COX) limits the vasodilatation, and (2) COX contributes to β‐adrenergic sweating. In 10 young males (25 ± 5 years), cutaneous vascular conductance (CVC) and sweat rate were evaluated at four intradermal forearm skin sites infused with (1) lactated Ringer solution (control), (2) 10 mm Nω‐nitro‐l‐arginine (l‐NNA), a non‐specific NOS inhibitor, (3) 10 mm ketorolac, a non‐specific COX inhibitor, or (4) a combination of l‐NNA and ketorolac. All sites were co‐administered with a high dose of isoproterenol (100 μm) for 3 min to maximally induce β‐adrenergic sweating (β‐adrenergic sweating is significantly blunted by subsequent activations). Approximately 60 min after the washout period, three incremental doses of isoproterenol were co‐administered (1, 10 and 100 μm each for 25 min). Increases in CVC induced by the first and second 100 μm isoproterenol were attenuated by l‐NNA alone, and those in response to all doses of isoproterenol were reduced by l‐NNA with co‐infusion of ketorolac (all P ≤ 0.05). Ketorolac alone augmented increases in CVC induced by 10 μm and by the second 100 μm isoproterenol (both P ≤ 0.05). While isoproterenol‐induced sweating was not affected by the separate administration of l‐NNA or ketorolac (all P > 0.05), their combined administration augmented sweating elicited by the first 3 min of 100 μm isoproterenol (P = 0.05). We show that while NOS contributes to β‐adrenergic cutaneous vasodilatation, COX restrains the vasodilatation. Finally, combined inhibition of NOS and COX augments β‐adrenergic sweating.

Key points
Muscle atrophy is a debilitating condition that affects a high percentage of the population with a negative impact on quality of life.
Dissecting the molecular level of the atrophy process, and the similarities/dissimilarities among different catabolic conditions, is a necessary step for designing specific countermeasures to attenuate/prevent muscle loss.
The FoxO family transcription factors represent one of the most important regulators of atrophy programme stimulating the expression of many atrophy‐related genes.
The findings of the present study clearly indicate that the signalling network controlling the atrophy programme is specific for each catabolic condition.
Abstract
Muscle atrophy is a complex process that is in common with many different catabolic diseases including disuse/inactivity and ageing. The signalling pathways that control the atrophy programme in the different disuse/inactivity conditions have not yet been completely dissected. The inhibition of FoxO is considered to only partially spare muscle mass after denervation. The present study aimed: (i) to determine the involvement of FoxOs in hindlimb suspension disuse model; (ii) to define whether the molecular events of protein breakdown are shared among different unloaded muscles; and finally (iii) to compare the data obtained in this model with another model of inactivity such as denervation. Both wild‐type and muscle‐specific FoxO1,3,4 knockout (FoxO1,3,4−/−) mice were unloaded for 3 and 14 days and muscles were characterized by functional, morphological, biochemical and molecular assays. The data obtained show that FoxOs are required for muscle loss and force drop during unloading. Moreover, we found that FoxO‐dependent atrogenes vary in different unloaded muscles and that they diverge from denervation. The findings of the present study clearly indicate that the signalling network that controls the atrophy programme is specific for each catabolic condition.

Key points
Distinct Ca2+ channels work in a coordinated manner to grade Ca2+ spark/spontaneous transient outward currents (STOCs) in rat cerebral arteries.
The relative contribution of each Ca2+ channel to Ca2+ spark/STOC production depends upon their biophysical properties and the resting membrane potential of smooth muscle.
Na+/Ca2+ exchanger, but not TRP channels, can also facilitate STOC production.
Abstract
Ca2+ sparks are generated in a voltage‐dependent manner to initiate spontaneous transient outward currents (STOCs), events that moderate arterial constriction. In this study, we defined the mechanisms by which membrane depolarization increases Ca2+ sparks and subsequent STOC production. Using perforated patch clamp electrophysiology and rat cerebral arterial myocytes, we monitored STOCs in the presence and absence of agents that modulate Ca2+ entry. Beginning with CaV3.2 channel inhibition, Ni2+ was shown to decrease STOC frequency in cells held at hyperpolarized (−40 mV) but not depolarized (−20 mV) voltages. In contrast, nifedipine, a CaV1.2 inhibitor, markedly suppressed STOC frequency at −20 mV but not −40 mV. These findings aligned with the voltage‐dependent profiles of L‐ and T‐type Ca2+ channels. Furthermore, computational and experimental observations illustrated that Ca2+ spark production is intimately tied to the activity of both conductances. Intriguingly, this study observed residual STOC production at depolarized voltages that was independent of CaV1.2 and CaV3.2. This residual component was insensitive to TRPV4 channel modulation and was abolished by Na+/Ca2+ exchanger blockade. In summary, our work highlights that the voltage‐dependent triggering of Ca2+ sparks/STOCs is not tied to a single conductance but rather reflects an interplay among multiple Ca2+ permeable pores with distinct electrophysiological properties. This integrated orchestration enables smooth muscle to grade Ca2+ spark/STOC production and thus precisely tune negative electrical feedback.

Key points
Myosin filament mechanosensing determines the efficiency of the contraction by adapting the number of switched ON motors to the load.
Accordingly, the unloaded shortening velocity (V0) is already set at the end of latency relaxation (LR), ∼10 ms after the start of stimulation, when the myosin filament is still in the OFF state.
Here the number of actin‐attached motors per half‐myosin filament (n) during V0 shortening imposed either at the end of LR or at the plateau of the isometric contraction is estimated from the relation between half‐sarcomere compliance and force during the force redevelopment after shortening.
The value of n decreases progressively with shortening and, during V0 shortening starting at the end of LR, is 1–4.
Reduction of n is accounted for by a constant duty ratio of 0.05 and a parallel switching OFF of motors, explaining the very low rate of ATP utilization found during unloaded shortening.
Abstract
The maximum velocity at which a skeletal muscle can shorten (i.e. the velocity of sliding between the myosin filament and the actin filament under zero load, V0) is already set at the end of the latency relaxation (LR) preceding isometric force generation, ∼10 ms after the start of electrical stimulation in frog muscle fibres at 4°C. At this time, Ca2+‐induced activation of the actin filament is maximal, while the myosin filament is in the OFF state characterized by most of the myosin motors lying on helical tracks on the filament surface, making them unavailable for actin binding and ATP hydrolysis. Here, the number of actin‐attached motors per half‐thick filament during V0 shortening (n) is estimated by imposing, on tetanized single fibres from Rana esculenta (at 4°C and sarcomere length 2.15 μm), small 4 kHz oscillations and determining the relation between half‐sarcomere (hs) compliance and force during the force development following V0 shortening. When V0 shortening is superimposed on the maximum isometric force T0, n decreases progressively with the increase of shortening (range 30–80 nm per hs) and, when V0 shortening is imposed at the end of LR, n can be as low as 1–4. Reduction of n is accounted for by a constant duty ratio of the myosin motor of ∼0.05 and a parallel switching OFF of the thick filament, providing an explanation for the very low rate of ATP utilization during extended V0 shortening.

Key points
Neuromuscular acetylcholine (ACh) receptors have a high affinity for the neurotransmitter ACh and a low affinity for its metabolic product choline.
At each transmitter binding site three aromatic groups determine affinity, and together provide ∼50% more binding energy for ACh than for choline.
Deprotonation of αY190 by a nearby lysine strengthens the interaction between this aromatic ring and both ACh and choline.
H‐bonds position ACh and choline differently in the aromatic cage to generate the different affinities.
Abstract
Acetylcholine (ACh) released at the vertebrate nerve‐muscle synapse is hydrolysed rapidly to choline (Cho), so endplate receptors (AChRs) are exposed to high concentrations of both of these structurally related ligands. To understand how these receptors distinguish ACh and Cho, we used single‐channel electrophysiology to measure resting affinities (binding free energies) of these and other agonists in adult‐type mouse AChRs having a mutation(s) at the transmitter‐binding sites. The aromatic rings of αY190, αW149 and αY198 each provide ∼50% less binding energy for Cho compared to ACh. At αY198 a phenylalanine substitution had no effect, but at αY190 this substitution caused a large, agonist‐independent loss in binding energy that depended on the presence of αK145. The results suggest that (1) αY190 is deprotonated by αK145 to strengthen the interaction between this benzene ring and the agonist's quaternary ammonium (QA) and (2) AChRs respond strongly to ACh because an H‐bond positions the QA to interact optimally with the rings, and weakly to Cho because a different H‐bond tethers the ligand to misalign the QA and form weaker interactions with the aromatic groups. The results suggest that the difference in ACh versus Cho binding energies is determined by different ligand positions within a fixed protein structure.

Key Points
Sensory gating is important for preventing excessive environmental stimulation from overloading neural resources.
Gating in the human somatosensory cortices is a critically understudied topic, particularly in the lower extremities.
We utilize the unique capabilities of magnetoencephalographic neuroimaging to quantify the normative neural population responses and dynamic functional connectivity of somatosensory gating in the lower extremities of healthy human participants.
We show that somatosensory processing is subserved by a robust gating effect in the oscillatory domain, as well as a dynamic effect on interhemispheric functional connectivity between primary sensory cortices.
These results provide novel insight into the dynamic neural mechanisms that underlie the processing of somatosensory information in the human brain, and will be vital in better understanding the neural responses that are aberrant in gait‐related neurological disorders (e.g. cerebral palsy).
Abstract
Sensory gating (SG) is a phenomenon in which neuronal responses to subsequent similar stimuli are weaker, and is considered to be an important mechanism for preventing excessive environmental stimulation from overloading shared neural resources. Although gating has been demonstrated in multiple sensory systems, the neural dynamics and developmental trajectory underlying SG remain poorly understood. In the present study, we adopt a data‐driven approach to map the spectrotemporal amplitude and functional connectivity (FC) dynamics that support gating in the somatosensory system (somato‐SG) in healthy children and adolescents using magnetoencephalography (MEG). These data underwent time‐frequency decomposition and the significant signal changes were imaged using a beamformer. Voxel time series were then extracted from the peak voxels and these signals were examined in the time and time‐frequency domains, and then subjected to dynamic FC analysis. The results obtained indicate a significant decrease in the amplitude of the neural response following the second stimulation relative to the first in the primary somatosensory cortex (SI). A significant decrease in response latency was also found between stimulations, and each stimulation induced a sharp decrease in FC between somatosensory cortical areas. Furthermore, there were no significant correlations between somato‐SG metrics and age. We conclude that somato‐SG can be observed in SI in both the time and oscillatory domains, with rich dynamics and alterations in inter‐hemispheric FC, and that this phenomenon has already matured by early childhood. A better understanding of these dynamics may provide insight to the numerous psychiatric and neurologic conditions that have been associated with aberrant SG across multiple modalities.

Key points
This study investigates the effects of cholinergic transmission on the expiratory oscillator, the parafacial respiratory group (pFRG) in urethane anaesthetized adult rats.
Local inhibition of the acetyl cholinesterase enzyme induced activation of expiratory abdominal muscles and active expiration.
Local application of the cholinomimetic carbachol elicited recruitment of late expiratory neurons, expiratory abdominal muscle activity and active expiration. This effect was antagonized by local application of the muscarinic antagonists scopolamine, J104129 and 4DAMP.
We observed distinct physiological responses between the more medial chemosensitive region of the retrotrapezoid nucleus and the more lateral region of pFRG.
These results support the hypothesis that pFRG is under cholinergic neuromodulation and the region surrounding the facial nucleus contains a group of neurons with distinct physiological roles.
Abstract
Active inspiration and expiration are opposing respiratory phases generated by two separate oscillators in the brainstem: inspiration driven by a neuronal network located in the preBötzinger complex (preBötC) and expiration driven by a neuronal network located in the parafacial respiratory group (pFRG). While continuous activity of the preBötC is necessary for maintaining ventilation, the pFRG behaves as a conditional expiratory oscillator, being silent in resting conditions and becoming rhythmically active in the presence of increased respiratory drive (e.g. hypoxia, hypercapnia, exercise and through release of inhibition). Recent evidence from our laboratory suggests that expiratory activity in the principal expiratory pump muscles, the abdominals, is modulated in a state‐dependent fashion, frequently occurring during periods of REM sleep. We hypothesized that acetylcholine, a neurotransmitter released in wakefulness and REM sleep by mesopontine structures, contributes to the activation of pFRG neurons and thus acts to promote the recruitment of expiratory abdominal muscle activity. We investigated the stimulatory effect of cholinergic neurotransmission on pFRG activity and recruitment of active expiration in vivo under anaesthesia. We demonstrate that local application of the acetylcholinesterase inhibitor physostigmine into the pFRG potentiated expiratory activity. Furthermore, local application of the cholinomimetic carbachol into the pFRG activated late expiratory neurons and induced long lasting rhythmic active expiration. This effect was completely abolished by pre‐application of the muscarinic antagonist scopolamine, and more selective M3 antagonists 4DAMP and J104129. We conclude that cholinergic muscarinic transmission contributes to excitation of pFRG neurons and promotes both active recruitment of abdominal muscles and active expiratory flow.

Key points
Common complications of pregnancy, such as chronic fetal hypoxia, trigger a fetal origin of cardiovascular dysfunction and programme cardiovascular disease in later life.
Sildenafil treatment protects placental perfusion and fetal growth, but whether the effects of sildenafil transcend the placenta to affect the fetus is unknown.
Using the chick embryo model, here we show that sildenafil treatment directly protects the fetal cardiovascular system in hypoxic development, and that the mechanisms of sildenafil protection include reduced oxidative stress and increased nitric oxide bioavailability;
Sildenafil does not protect against fetal growth restriction in the chick embryo, supporting the idea that the protective effect of sildenafil on fetal growth reported in mammalian studies, including humans, is secondary to improved placental perfusion.
Therefore, sildenafil may be a good candidate for human translational antioxidant therapy to protect the chronically hypoxic fetus in adverse pregnancy.
Abstract
There is a need for developing clinically translatable therapy for preventing fetal origins of cardiovascular disease in pregnancy complicated by chronic fetal hypoxia. Evidence shows that sildenafil protects placental perfusion and fetal growth. However, whether beneficial effects of sildenafil transcend onto the fetal heart and circulation in complicated development is unknown. We isolated the direct effects of sildenafil on the fetus using the chick embryo and hypothesised that sildenafil also protects fetal cardiovascular function in hypoxic development. Chick embryos (n = 11 per group) were incubated in normoxia or hypoxia (14% O2) from day 1 and treated with sildenafil (4 mg kg−1 day−1) from day 13 of the 21‐day incubation. Hypoxic incubation increased oxidative stress (4‐hydroxynonenal, 141.1 ± 17.6% of normoxic control), reduced superoxide dismutase (60.7 ± 6.3%), increased phosphodiesterase type 5 expression (167 ± 13.7%) and decreased nitric oxide bioavailability (54.7 ± 6.1%) in the fetal heart, and promoted peripheral endothelial dysfunction (70.9 ± 5.6% AUC of normoxic control; all P

Key points
Cerebral haemodynamic response to neural stimulation has been extensively investigated in animal and clinical studies, in both adult and paediatric populations, but little is known about cerebral haemodynamic functional response in the fetal brain.
The present study describes the cerebral haemodynamic response measured by near‐infrared spectroscopy to somatosensory stimulation in fetal sheep.
The cerebral haemodynamic response in the fetal sheep brain changes from a positive (increase in oxyhaemoglobin (oxyHb)) response pattern to a negative or biphasic response pattern when the duration of somatosensory stimulation is increased, probably due to cerebral vasoconstriction with prolonged stimulations.
In contrast to adult studies, we have found that changes in fetal cerebral blood flow and oxyHb are positively increased in response to somatosensory stimulation during hypercapnia. We propose this is related to reduced vascular resistance and recruitment of cerebral vasculature in the fetal brain during hypercapnia.
Abstract
Functional hyperaemia induced by a localised increase in neuronal activity has been suggested to occur in the fetal brain owing to a positive blood oxygen level‐dependent (BOLD) signal recorded by functional magnetic resonance imaging following acoustic stimulation. To study the effect of somatosensory input on local cerebral perfusion we used near‐infrared spectroscopy (NIRS) in anaesthetised, partially exteriorised fetal sheep where the median nerve was stimulated with trains of pulses (2 ms, 3.3 Hz) for durations of 1.8, 4.8 and 7.8 s. Signal averaging of cerebral NIRS responses to 20 stimulus trains repeated every 60 s revealed that a short duration of stimulation (1.8 s) increased oxyhaemoglobin in the contralateral cortex consistent with a positive functional response, whereas longer durations of stimulation (4.8, 7.8 s) produced more variable oxyhaemoglobin responses including positive, negative and biphasic patterns of change. Mean arterial blood pressure and cerebral perfusion as monitored by laser Doppler flowmetry always showed small, but coincident increases following median nerve stimulation regardless of the type of response detected by the NIRS in the contralateral cortex. Hypercapnia significantly increased the baseline total haemoglobin and deoxyhaemoglobin, and in 7 of 8 fetal sheep positively increased the changes in contralateral total haemoglobin and oxyhaemoglobin in response to the 7.8 s stimulus train, compared to the response recorded during normocapnia. These results show that activity‐driven changes in cerebral perfusion and oxygen delivery are present in the fetal brain, and persist even during periods of hypercapnia‐induced cerebral vasodilatation.

Key points
Fetal behavioural state in healthy late gestation pregnancy is affected by maternal position.
Fetal state 1F is more likely to occur in maternal supine or right lateral positions.
Fetal state 4F is less likely to occur when the woman lies supine or semi‐recumbent.
Fetal state change is more likely when the woman is supine or semi‐recumbent.
Fetal heart rate variability is affected by maternal position with variability reduced in supine and semi‐recumbent positions.
Abstract
Fetal behavioural states (FBS) are measures of fetal wellbeing. In acute hypoxaemia, the human fetus adapts to a lower oxygen consuming state with changes in the cardiotocograph and reduced fetal activity. Recent studies of late gestation stillbirth described the importance of sleep position in the risk of intrauterine death. We designed this study to assess the effects of different maternal positions on FBS in healthy late gestation pregnancies under controlled conditions. Twenty‐nine healthy women had continuous fetal ECG recordings under standardized conditions in four randomly allocated positions, left lateral, right lateral, supine and semi‐recumbent. Two blinded observers, assigned fetal states in 5 min blocks. Measures of fetal heart rate variability were calculated from ECG beat to beat data. Compared to state 2F, state 4F was less likely to occur when women were semi‐recumbent [odds ratio (OR) = 0.11, 95% confidence interval (95% CI) 0.02, 0.55], and supine (OR = 0.27, 95% CI 0.07, 1.10). State 1F was more likely on the right (OR = 2.36, 95% CI 1.11, 5.04) or supine (OR = 4.99, 95% CI 2.41, 10.43) compared to the left. State change was more likely when the mother was semi‐recumbent (OR = 2.17, 95% CI 1.19, 3.95) or supine (OR = 2.67, 95% CI 1.46, 4.85). There was a significant association of maternal position to mean fetal heart rate. The measures of heart rate variability (SDNN and RMSSD) were reduced in both semi‐recumbent and supine positions. In healthy late gestation pregnancy, maternal position affects FBS and heart rate variability. These effects are likely fetal adaptations to positions which may produce a mild hypoxic stress.

Key points
Ion channels are transmembrane proteins that are synthesized within the cells but need to be trafficked to the cell membrane for the channels to function.
Small‐conductance, Ca2+‐activated K+ channels (SK, KCa2) are unique subclasses of K+ channels that are regulated by Ca2+ inside the cells; they are expressed in human atrial myocytes and responsible for shaping atrial action potentials.
We have previously shown that interacting proteins of SK2 channels are important for channel trafficking to the membrane.
Using total internal reflection fluorescence (TIRF) and confocal microscopy, we studied the mechanisms by which the surface membrane localization of SK2 (KCa2.2) channels is regulated by their interacting proteins.
Understanding the mechanisms of SK channel trafficking may provide new insights into the regulation controlling the repolarization of atrial myocytes.
Abstract
The normal function of ion channels depends critically on the precise subcellular localization and the number of channel proteins on the cell surface membrane. Small‐conductance, Ca2+‐activated K+ channels (SK, KCa2) are expressed in human atrial myocytes and are responsible for shaping atrial action potentials. Understanding the mechanisms of SK channel trafficking may provide new insights into the regulation controlling the repolarization of atrial myocytes. We have previously demonstrated that the C‐ and N‐termini of SK2 channels interact with the actin‐binding proteins α‐actinin2 and filamin A, respectively. However, the roles of the interacting proteins on SK2 channel trafficking remain incompletely understood. Using total internal reflection fluorescence (TIRF) microscopy, we studied the mechanisms of surface membrane localization of SK2 (KCa2.2) channels. When SK2 channels were co‐expressed with filamin A or α‐actinin2, the membrane fluorescence intensity of SK2 channels increased significantly. We next tested the effects of primaquine and dynasore on SK2 channels expression. Treatment with primaquine significantly reduced the membrane expression of SK2 channels. In contrast, treatment with dynasore failed to alter the surface membrane expression of SK2 channels. Further investigations using constitutively active or dominant‐negative forms of Rab GTPases provided additional insights into the distinct roles of the two cytoskeletal proteins on the recycling processes of SK2 channels from endosomes. α‐Actinin2 facilitated recycling of SK2 channels from both early and recycling endosomes while filamin A probably aids the recycling of SK2 channels from recycling endosomes.

Key points
Humans are unique in controlling body temperature in a hot environment by a large amount of skin blood flow; however, the decrease in total peripheral resistance due to systemic cutaneous vasodilatation and the reduction of venous return to the heart due to blood pooling in the cutaneous vein threatens blood pressure maintenance in the upright position, and occasionally causes heat syncope.
Against this condition, cutaneous vasodilatation is reportedly suppressed to maintain arterial pressure; however, the nerve activity responsible for this phenomenon has not been identified.
In the present study, we found that the skin sympathetic nerve activity component that was synchronised with the cardiac cycle increased in hyperthermia, but the increase was suppressed when the posture was changed from supine to head‐up tilt.
The profile of the component agreed with that of cutaneous vasodilatation.
Thus, the component might contribute to the prevention of heat syncope in humans.
Abstract
In humans, the cutaneous vasodilatation response to hyperthermia has been suggested to be suppressed by baroreflexes to maintain arterial pressure when the posture is changed from supine to upright, and if the reflexes do not function sufficiently, it can cause heat syncope. However, the efferent signals of the reflexes have not been identified. To identify the signals, we continuously measured skin sympathetic nerve activity (SSNA; microneurography), right atrial volume (RAV; echocardiography, the baroreceptors for the reflexes are reportedly located in the right atrium), cutaneous vascular conductance on the chest (CVCchest; laser Doppler flowmetry), and oesophageal temperature (Toes; thermocouple) in young men before and after passive warming with a perfusion suit, during which periods the posture was changed from supine to 30 deg head‐up tilt positions. During these periods, we also simultaneously measured muscle sympathetic nerve activity (MSNA) to distinguish the SSNA from MSNA. We found that an increase in Toes by ∼0.7°C (P 0.26) although an increase in CVCchest (P

Key points
Intrauterine growth restriction (IUGR) is associated with vascular dysfunction, oxidative stress and signs of endothelial epigenetic programming of the umbilical vessels.
There is no evidence that this epigenetic programming is occurring on systemic fetal arteries.
In IUGR guinea pigs we studied the functional and epigenetic programming of endothelial nitric oxide synthase (eNOS) (Nos3 gene) in umbilical and systemic fetal arteries, addressing the role of oxidative stress in this process by maternal treatment with N‐acetylcysteine (NAC) during the second half of gestation.
The present study suggests that IUGR endothelial cells have common molecular markers of programming in umbilical and systemic arteries. Notably, maternal treatment with NAC restores fetal growth by increasing placental efficiency and reverting the functional and epigenetic programming of eNOS in arterial endothelium in IUGR guinea pigs.
Abstract
In humans, intrauterine growth restriction (IUGR) is associated with vascular dysfunction, oxidative stress and signs of endothelial programming in umbilical vessels. We aimed to determine the effects of maternal antioxidant treatment with N‐acetylcysteine (NAC) on fetal endothelial function and endothelial nitric oxide synthase (eNOS) programming in IUGR guinea pigs. IUGR was induced by implanting ameroid constrictors on uterine arteries of pregnant guinea pigs at mid gestation, half of the sows receiving NAC in the drinking water (from day 34 until term). Fetal biometry and placental vascular resistance were followed by ultrasound throughout gestation. At term, umbilical arteries and fetal aortae were isolated to assess endothelial function by wire‐myography. Primary cultures of endothelial cells (ECs) from fetal aorta, femoral and umbilical arteries were used to determine eNOS mRNA levels by quantitative PCR and analyse DNA methylation in the Nos3 promoter by pyrosequencing. Doppler ultrasound measurements showed that NAC reduced placental vascular resistance in IUGR (P

Key points
Intracellular Na+‐activated Slo2 potassium channels are in a closed state under normal physiological conditions, although their mechanisms of ion permeation gating are not well understood.
A cryo‐electron microscopy structure of Slo2.2 suggests that the ion permeation pathway of these channels is closed by a single constriction of the inner pore formed by the criss‐crossing of the cytoplasmic ends of the S6 segments (the S6 bundle crossing) at a conserved Met residue.
Functional characterization of mutant Slo2 channels suggests that hydrophobic interactions between Leu residues in the upper region of the S6 segments contribute to stabilizing the inner pore in a non‐conducting state.
Mutation of the conserved Met residues in the S6 segments to the negatively‐charged Glu did not induce constitutive opening of Slo2.1 or Slo2.2, suggesting that ion permeation of Slo2 channels is not predominantly gated by the S6 bundle crossing.
Abstract
Large conductance K+‐selective Slo2 channels are in a closed state unless activated by elevated [Na+]i. Our previous studies suggested that the pore helix/selectivity filter serves as the activation gate in Slo2 channels. In the present study, we evaluated two other potential mechanisms for stabilization of Slo2 channels in a closed state: (1) dewetting and collapse of the inner pore (hydrophobic gating) and (2) constriction of the inner pore by tight criss‐crossing of the cytoplasmic ends of the S6 α‐helical segments. Slo2 channels contain two conserved Leu residues in each of the four S6 segments that line the inner pore region nearest the bottom of the selectivity filter. To evaluate the potential role of these residues in hydrophobic gating, Leu267 and Leu270 in human Slo2.1 were each replaced by 15 different residues. The relative conductance of mutant channels was highly dependent on hydrophilicity and volume of the amino acid substituted for Leu267 and was maximal with L267H. Consistent with their combined role in hydrophobic gating, replacement of both Leu residues with the isosteric but polar residue Asn (L267N/L270N) stabilized channels in a fully open state. In a recent cryo‐electron microscopy structure of chicken Slo2.2, the ion permeation pathway of the channel is closed by a constriction of the inner pore formed by criss‐crossing of the S6 segments at a conserved Met. Inconsistent with the S6 segment crossing forming the activation gate, replacement of the homologous Met residues in human Slo2.1 or Slo2.2 with the negatively‐charged Glu did not induce constitutive channel opening.

Key points
Synaptic transmission is mediated by the release of neurotransmitters from synaptic vesicles in response to stimulation or through the spontaneous fusion of a synaptic vesicle with the presynaptic plasma membrane.
There is growing evidence that synaptic vesicles undergoing spontaneous fusion versus those fusing in response to stimuli are functionally distinct.
In this study, we acutely probe the effects of intravesicular free radical generation on synaptic vesicles that fuse spontaneously or in response to stimuli.
By targeting vesicles that preferentially release spontaneously, we can dissociate the effects of intravesicular free radical generation on spontaneous neurotransmission from evoked neurotransmission and vice versa.
Taken together, these results further advance our knowledge of the synapse and the nature of the different synaptic vesicle pools mediating neurotransmission.
Abstract
Earlier studies suggest that spontaneous and evoked neurotransmitter release processes are maintained by synaptic vesicles which are segregated into functionally distinct pools. However, direct interrogation of the link between this putative synaptic vesicle pool heterogeneity and neurotransmission has been difficult. To examine this link, we tagged vesicles with horseradish peroxidase (HRP) – a haem‐containing plant enzyme – or antibodies against synaptotagmin‐1 (syt1). Filling recycling vesicles in hippocampal neurons with HRP and subsequent treatment with hydrogen peroxide (H2O2) modified the properties of neurotransmitter release depending on the route of HRP uptake. While strong depolarization‐induced uptake of HRP suppressed evoked release and augmented spontaneous release, HRP uptake during mild activity selectively impaired evoked release, whereas HRP uptake at rest solely potentiated spontaneous release. Expression of a luminal HRP‐tagged syt1 construct and subsequent H2O2 application resulted in a similar increase in spontaneous release and suppression as well as desynchronization of evoked release, recapitulating the canonical syt1 loss‐of‐function phenotype. An antibody targeting the luminal domain of syt1, on the other hand, showed that augmentation of spontaneous release and suppression of evoked release phenotypes are dissociable depending on whether the antibody uptake occurred at rest or during depolarization. Taken together, these findings indicate that vesicles that maintain spontaneous and evoked neurotransmitter release preserve their identity during recycling and syt1 function in suppression of spontaneous neurotransmission can be acutely dissociated from syt1 function to synchronize synaptic vesicle exocytosis upon stimulation.

Key points
Auditory brainstem neurons involved in sound source localization are equipped with several morphological and molecular features that enable them to compute interaural level and time differences.
As sound source localization works continually, synaptic transmission between these neurons should be reliable and temporally precise, even during sustained periods of high‐frequency activity.
Using patch‐clamp recordings in acute brain slices, we compared synaptic reliability and temporal precision in the seconds–minute range between auditory and two types of hippocampal synapses; the latter are less confronted with temporally precise high‐frequency transmission than the auditory ones.
We found striking differences in synaptic properties (e.g. continually high quantal content) that allow auditory synapses to reliably release vesicles at much higher rate than their hippocampal counterparts.
Thus, they are indefatigable and also in a position to transfer information with exquisite temporal precision and their performance appears to be supported by very efficient replenishment mechanisms.
Abstract
At early stations of the auditory pathway, information is encoded by precise signal timing and rate. Auditory synapses must maintain the relative timing of events with submillisecond precision even during sustained and high‐frequency stimulation. In non‐auditory brain regions, e.g. telencephalic ones, synapses are activated at considerably lower frequencies. Central to understanding the heterogeneity of synaptic systems is the elucidation of the physical, chemical and biological factors that determine synapse performance. In this study, we used slice recordings from three synapse types in the mouse auditory brainstem and hippocampus. Whereas the auditory brainstem nuclei experience high‐frequency activity in vivo, the hippocampal circuits are activated at much lower frequencies. We challenged the synapses with sustained high‐frequency stimulation (up to 200 Hz for 60 s) and found significant performance differences. Our results show that auditory brainstem synapses differ considerably from their hippocampal counterparts in several aspects, namely resistance to synaptic fatigue, low failure rate and exquisite temporal precision. Their high‐fidelity performance supports the functional demands and appears to be due to the large size of the readily releasable pool and a high release probability, which together result in a high quantal content. In conjunction with very efficient vesicle replenishment mechanisms, these properties provide extremely rapid and temporally precise signalling required for neuronal communication at early stations of the auditory system, even during sustained activation in the minute range.

Key points
Increase in blood pressure in the renal afferent arteriole is known to induce an increase in cytosolic calcium concentration ([Ca2+]i) of juxtaglomerular (JG) cells and to result in a decreased secretion of renin.
Mechanical stimulation of As4.1 JG cells induces an increase in [Ca2+]i that is inhibited by HC067047 and RN1734, two inhibitors of TRPV4, or by siRNA‐mediated repression of TRPV4.
Inhibition of TRPV4 impairs pressure‐induced decrease in renin secretion.
Compared to wild‐type mice, Trpv4−/− mice present increased resting plasma levels of renin and aldosterone and present a significantly altered pressure–renin relationship.
We suggest that TRPV4 channel participates in mechanosensation at the juxtaglomerular apparatus.
Abstract
The renin–angiotensin system is a crucial blood pressure regulation system. It consists of a hormonal cascade where the rate‐limiting enzyme is renin, which is secreted into the blood flow by renal juxtaglomerular (JG) cells in response to low pressure in the renal afferent arteriole. In contrast, an increase in blood pressure results in a decreased renin secretion. This is accompanied by a transitory increase in [Ca2+]i of JG cells. The inverse relationship between [Ca2+]i and renin secretion has been called the ‘calcium paradox’ of renin release. How increased pressure induces a [Ca2+]i transient in JG cells, is however, unknown. We observed that [Ca2+]i transients induced by mechanical stimuli in JG As4.1 cells were completely abolished by HC067047 and RN1734, two inhibitors of TRPV4. They were also reduced by half by siRNA‐mediated repression of TRPV4 but not after repression or inhibition of TRPV2 or Piezo1 ion channels. Interestingly, the stimulation of renin secretion by the adenylate cyclase activator forskolin was totally inhibited by cyclic stretching of the cells. This effect was mimicked by stimulation with GSK1016790A and 4αPDD, two activators of TRPV4 and inhibited in the presence of HC067047. Moreover, in isolated perfused kidneys from Trpv4−/− mice, the pressure–renin relationship was significantly altered. In vivo, Trpv4−/− mice presented increased plasma levels of renin and aldosterone compared to wild‐type mice. Altogether, our results suggest that TRPV4 is involved in the pressure‐induced entry of Ca2+ in JG cells, which inhibits renin release and allows the negative feedback regulation on blood pressure.

Key points
Altered cerebral autoregulation (CA) in obstructive sleep apnoea (OSA) patients may contribute to increased stroke risk in this population; the gold standard treatment for OSA is continuous positive airway pressure, which improves cerebrovascular regulation and may decrease the risk of stroke.
Isocapnic‐hypoxia impairs CA in healthy subjects, but it remains unknown in OSA whether impaired CA is further exacerbated by isocapnic‐hypoxia and whether it is improved by treatment with continuous positive airway pressure.
During normoxia, CA was altered in the more severe but not in the less severe OSA patients, while, in contrast, during isocapnic‐hypoxia, CA was similar between groups and tended to improve in patients with more severe OSA compared to normoxia.
From a clinical perspective, one month of continuous positive airway pressure treatment does not improve CA.
From a physiological perspective, this study suggests that sympathetic overactivity may be responsible for altered CA in the more severe OSA patients.
Abstract
Cerebral autoregulation (CA) impairment may contribute to the increased risk of stroke associated with obstructive sleep apnoea (OSA). It is unknown if impaired CA is further exacerbated by isocapnic‐hypoxia and whether it is improved by treatment of OSA with continuous positive airway pressure (CPAP). CA was assessed during wakefulness in 53 OSA patients (50.3 ± 9.3 years) and 21 controls (49.8 ± 8.6 years) at baseline and following a minimum of 1 month of effective CPAP therapy (OSA patients, n = 40). Control participants (n = 21) performed a follow‐up visit to control for time effects within OSA patients between baseline and the post‐CPAP visit. Beat‐by‐beat middle cerebral artery blood flow velocity and mean arterial blood pressure (MBP), and breath‐by‐breath end‐tidal partial pressure of CO2 (P ET ,CO2) were monitored. CA was determined during normoxia and isocapnic‐hypoxia using transfer function (phase and gain) and coherence analysis (including multiple and partial coherence (using MBP and P ET ,CO2 as inputs)) in the very low frequency range (0.03–0.07 Hz). OSA patients were divided into two subgroups (less severe and more severe) based upon the median respiratory disturbance index (RDI). During normoxia, the more severe OSA patients (RDI 45.9 ± 10.3) exhibited altered CA compared to controls and the less severe OSA patients (RDI 24.5 ± 5.9). In contrast, during isocapnic‐hypoxia, CA was similar between groups. CPAP had no effect on CA. In conclusion, CA is altered in the more severe OSA patients during normoxia but not during isocapnic‐hypoxia and CPAP treatment does not impact CA.

Key points
Vascular brain lesions and atherosclerosis are two similar conditions that are characterized by increased inflammation and oxidative stress.
Non‐invasive imaging in a murine model of atherosclerosis showed vascular brain damage and peripheral inflammation.
In this study, exercise training reduced magnetic resonance imaging‐detected abnormalities, insulin resistance and markers of oxidative stress and inflammation in old ApoE−/− mice.
Our results demonstrate the protective effect of exercise on neurovascular damage in the ageing brain of ApoE−/− mice.
Abstract
Vascular brain lesions, present in advanced atherosclerosis, share pathological hallmarks with peripheral vascular lesions, such as increased inflammation and oxidative stress. Physical activity reduces these peripheral risk factors, but its cerebrovascular effect is less documented, especially by non‐invasive imaging. Through a combination of in vivo and post‐mortem techniques, we aimed to characterize vascular brain damage in old ApoE−/− mice fed a high‐cholesterol (HC) diet with dietary controlled intake. We then sought to determine the beneficial effects of exercise training on oxidative stress and inflammation in the brain as a treatment option in an ageing atherosclerosis mouse model. Using in vivo magnetic resonance imaging (MRI) and biological markers of oxidative stress and inflammation, we evaluated the occurrence of vascular abnormalities in the brain of HC‐diet fed ApoE−/− mice >70 weeks old, its association with local and systemic oxidative stress and inflammation, and whether both can be modulated by exercise. Exercise training significantly reduced both MRI‐detected abnormalities (present in 71% of untrained vs. 14% of trained mice) and oxidative stress (lipid peroxidation, 9.1 ± 1.4 vs. 5.2 ± 0.9 μmol mg−1; P

Key points
Transcriptional co‐activator PGC‐1α1 has been shown to regulate energy metabolism and to mediate metabolic adaptations in pathological and physiological cardiac hypertrophy but other functional implications of PGC‐1α1 expression are not known.
Transgenic PGC‐1α1 overexpression within the physiological range in mouse heart induces purposive changes in contractile properties, electrophysiology and calcium signalling but does not induce substantial metabolic remodelling.
The phenotype of the PGC‐1α1 transgenic mouse heart recapitulates most of the functional modifications usually associated with the exercise‐induced heart phenotype, but does not protect the heart against load‐induced pathological hypertrophy.
Transcriptional effects of PGC‐1α1 show clear dose‐dependence with diverse changes in genes in circadian clock, heat shock, excitability, calcium signalling and contraction pathways at low overexpression levels, while metabolic genes are recruited at much higher PGC‐1α1 expression levels.
These results imply that the physiological role of PGC‐1α1 is to promote a beneficial excitation–contraction coupling phenotype in the heart.
Abstract
The transcriptional coactivator PGC‐1α1 has been identified as a central factor mediating metabolic adaptations of the heart. However, to what extent physiological changes in PGC‐1α1 expression levels actually contribute to the functional adaptation of the heart is still mostly unresolved. The aim of this study was to characterize the transcriptional and functional effects of physiologically relevant, moderate PGC‐1α1 expression in the heart. In vivo and ex vivo physiological analysis shows that expression of PGC‐1α1 within a physiological range in mouse heart does not induce the expected metabolic alterations, but instead induces a unique excitation–contraction (EC) coupling phenotype recapitulating features typically seen in physiological hypertrophy. Transcriptional screening of PGC‐1α1 overexpressing mouse heart and myocyte cultures with higher, acute adenovirus‐induced PGC‐1α1 expression, highlights PGC‐1α1 as a transcriptional coactivator with a number of binding partners in various pathways (such as heat shock factors and the circadian clock) through which it acts as a pleiotropic transcriptional regulator in the heart, to both augment and repress the expression of its target genes in a dose‐dependent fashion. At low levels of overexpression PGC‐1α1 elicits a diverse transcriptional response altering the expression state of circadian clock, heat shock, excitability, calcium signalling and contraction pathways, while metabolic targets of PGC‐1α1 are recruited at higher PGC‐1α1 expression levels. Together these findings demonstrate that PGC‐1α1 elicits a dual effect on cardiac transcription and phenotype. Further, our results imply that the physiological role of PGC‐1α1 is to promote a beneficial EC coupling phenotype in the heart.

Key points
We establish experimental preparations for optogenetic investigation of glutamatergic input to the inferior olive.
Neurones in the principal olivary nucleus receive monosynaptic extra‐somatic glutamatergic input from the neocortex.
Glutamatergic inputs to neurones in the inferior olive generate bidirectional postsynaptic potentials (PSPs), with a fast excitatory component followed by a slower inhibitory component.
Small conductance calcium‐activated potassium (SK) channels are required for the slow inhibitory component of glutamatergic PSPs and oppose temporal summation of inputs at intervals ≤ 20 ms.
Active integration of synaptic input within the inferior olive may play a central role in control of olivo‐cerebellar climbing fibre signals.
Abstract
The inferior olive plays a critical role in motor coordination and learning by integrating diverse afferent signals to generate climbing fibre inputs to the cerebellar cortex. While it is well established that climbing fibre signals are important for motor coordination, the mechanisms by which neurones in the inferior olive integrate synaptic inputs and the roles of particular ion channels are unclear. Here, we test the hypothesis that neurones in the inferior olive actively integrate glutamatergic synaptic inputs. We demonstrate that optogenetically activated long‐range synaptic inputs to the inferior olive, including projections from the motor cortex, generate rapid excitatory potentials followed by slower inhibitory potentials. Synaptic projections from the motor cortex preferentially target the principal olivary nucleus. We show that inhibitory and excitatory components of the bidirectional synaptic potentials are dependent upon AMPA (GluA) receptors, are GABAA independent, and originate from the same presynaptic axons. Consistent with models that predict active integration of synaptic inputs by inferior olive neurones, we find that the inhibitory component is reduced by blocking large conductance calcium‐activated potassium channels with iberiotoxin, and is abolished by blocking small conductance calcium‐activated potassium channels with apamin. Summation of excitatory components of synaptic responses to inputs at intervals ≤ 20 ms is increased by apamin, suggesting a role for the inhibitory component of glutamatergic responses in temporal integration. Our results indicate that neurones in the inferior olive implement novel rules for synaptic integration and suggest new principles for the contribution of inferior olive neurones to coordinated motor behaviours.

Key points
Research indicates that individuals may experience a rise (positive responders) or fall (negative responders) in muscle sympathetic nerve activity (MSNA) during mental stress.
In this study, we examined the early blood pressure responses (including the peak, time of peak and rate of rise in blood pressure) to mental stress in positive and negative responders.
Negative MSNA responders to mental stress exhibit a more rapid rise in diastolic pressure at the onset of the stressor, suggesting a baroreflex‐mediated suppression of MSNA. In positive responders there is a more sluggish rise in blood pressure during mental stress, which appears to be MSNA‐driven.
This study suggests that whether MSNA has a role in the pressor response is dependent upon the reactivity of blood pressure early in the task.
Abstract
Research indicates that individuals may experience a rise (positive responders) or fall (negative responders) in muscle sympathetic nerve activity (MSNA) during mental stress. The aim was to examine the early blood pressure response to stress in positive and negative responders and thus its influence on the direction of change in MSNA. Blood pressure and MSNA were recorded continuously in 21 healthy young males during 2 min mental stressors (mental arithmetic, Stroop test) and physical stressors (cold pressor, handgrip exercise, post‐exercise ischaemia). Participants were classified as negative or positive responders according to the direction of the mean change in MSNA during the stressor tasks. The peak changes, time of peak and rate of changes in blood pressure were compared between groups. During mental arithmetic negative responders experienced a significantly greater rate of rise in diastolic blood pressure in the first minute of the task (1.3 ± 0.5 mmHg s−1) compared with positive responders (0.4 ± 0.1 mmHg s−1; P = 0.03). Similar results were found for the Stroop test. Physical tasks elicited robust parallel increases in blood pressure and MSNA across participants. It is concluded that negative MSNA responders to mental stress exhibit a more rapid rise in diastolic pressure at the onset of the stressor, suggesting a baroreflex‐mediated suppression of MSNA. In positive responders there is a more sluggish rise in blood pressure during mental stress, which appears to be MSNA‐driven. This study suggests that whether MSNA has a role in the pressor response is dependent upon the reactivity of blood pressure early in the task.

Key points
Understanding how skeletal muscles respond to high temperatures may help develop strategies for improving exercise tolerance and preventing heat injury.
Mitochondria regulate cell survival by constantly changing their morphology through fusion and fission in response to environmental stimuli. Little is known about the involvement of mitochondrial dynamics in tolerance of skeletal muscle against heat stress.
Mild heat acclimation and moderate heat shock appear to have different effects on the mitochondrial morphology and fission protein Drp1 in skeletal muscle cells. Mitochondrial integrity plays a key role in cell survival under heat stress.
Abstract
The regulation of mitochondrial morphology is closely coupled to cell survival during stress. We examined changes in the mitochondrial morphology of mouse C2C12 skeletal muscle cells in response to heat acclimation and heat shock exposure. Acclimated cells showed a greater survival rate during heat shock exposure than non‐acclimated cells, and were characterized by long interconnected mitochondria and reduced expression of dynamin‐related protein 1 (Drp1) for their mitochondrial fractions. Exposure of C2C12 muscle cells to heat shock led to apoptotic death featuring activation of caspase 3/7, release of cytochrome c and loss of cell membrane integrity. Heat shock also caused excessive mitochondrial fragmentation, loss of mitochondrial membrane potential and production of reactive oxygen species in C2C12 cells. Western blot and immunofluorescence image analysis revealed translocation of Drp1 to mitochondria from the cytosol in C2C12 cells exposed to heat shock. Mitochondrial division inhibitor 1 or Drp1 gene silencer reduced mitochondrial fragmentation and increased cell viability during exposure to heat shock. These results suggest that Drp1‐dependent mitochondrial fission may regulate susceptibility to heat‐induced apoptosis in muscle cells and that Drp1 may serve as a target for the prevention of heat‐related injury.

Key points
It is controversial whether glutamate can leak out of vesicles in the nerve terminal.
To address this issue, we abolished vesicular glutamate uptake by washing out presynaptic cytosolic glutamate or by blocking vacuolar ATPase activity using bafilomycin A1.
In the absence of vesicular glutamate uptake, both spontaneous and nerve‐evoked EPSCs underwent a rundown, suggesting that vesicular glutamate can leak out of vesicles.
However, the rundown of evoked EPSCs was caused mainly by accumulation of unfilled vesicles after exocytic release of glutamate, suggesting a minor influence of glutamate leakage on synaptic transmission.
Abstract
Glutamate leaks out of synaptic vesicles when the transvesicular proton gradient is dissipated in isolated vesicle preparations. In the nerve terminal, however, it is controversial whether glutamate can leak out of vesicles. To address this issue, we abolished vesicular glutamate uptake by washing out presynaptic cytosolic glutamate in whole‐cell dialysis or by blocking vacuolar ATPase using bafilomycin A1 (Baf) at the calyx of Held in mouse brainstem slices. Presynaptic glutamate washout or Baf application reduced the mean amplitude and frequency of spontaneous miniature (m)EPSCs and the mean amplitude of EPSCs evoked every 10 min. The percentage reduction of mEPSC amplitude was much less than that of EPSC amplitude or mEPSC frequency, and tended to reach a plateau. The mean amplitude of mEPSCs after glutamate washout or Baf application remained high above the detection limit, deduced from the reduction of mEPSC amplitude by the AMPA receptor blocker 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione. Membrane capacitance measurements from presynaptic terminals indicated no effect of glutamate washout on exocytosis or endocytosis of synaptic vesicles. We conclude that glutamate can leak out of vesicles unless it is continuously taken up from presynaptic cytosol. However, the magnitude of glutamate leakage was small and had only a minor effect on synaptic responses. In contrast, prominent rundowns of EPSC amplitude and mEPSC frequency observed after glutamate washout or Baf application are likely to be caused by accumulation of unfilled vesicles in presynaptic terminals retrieved after spontaneous and evoked glutamate release.

Key points
Kv2 channels underlie delayed‐rectifier potassium currents in various neurons, although their physiological roles often remain elusive. Almost nothing is known about Kv2 channel functions in medial entorhinal cortex (mEC) neurons, which are involved in representing space, memory formation, epilepsy and dementia.
Stellate cells in layer II of the mEC project to the hippocampus and are considered to be space‐representing grid cells. We used the new Kv2 blocker Guangxitoxin‐1E (GTx) to study Kv2 functions in these neurons.
Voltage clamp recordings from mEC stellate cells in rat brain slices showed that GTx inhibited delayed‐rectifier K+ current but not transient A‐type current.
In current clamp, GTx had multiple effects: (i) increasing excitability and bursting at moderate spike rates but reducing firing at high rates; (ii) enhancing after‐depolarizations; (iii) reducing the fast and medium after‐hyperpolarizations; (iv) broadening action potentials; and (v) reducing spike clustering.
GTx is a useful tool for studying Kv2 channels and their functions in neurons.
Abstract
The medial entorhinal cortex (mEC) is strongly involved in spatial navigation, memory, dementia and epilepsy. Although potassium channels shape neuronal activity, their roles in mEC are largely unknown. We used the new Kv2 blocker Guangxitoxin‐1E (GTx; 10–100 nm) in rat brain slices to investigate Kv2 channel functions in mEC layer II stellate cells (SCs). These neurons project to the hippocampus and are considered to be grid cells representing space. Voltage clamp recordings from SCs nucleated patches showed that GTx inhibited a delayed rectifier K+ current activating beyond –30 mV but not transient A‐type current. In current clamp, GTx (i) had almost no effect on the first action potential but markedly slowed repolarization of late spikes during repetitive firing; (ii) enhanced the after‐depolarization (ADP); (iii) reduced fast and medium after‐hyperpolarizations (AHPs); (iv) strongly enhanced burst firing and increased excitability at moderate spike rates but reduced spiking at high rates; and (v) reduced spike clustering and rebound potentials. The changes in bursting and excitability were related to the altered ADPs and AHPs. Kv2 channels strongly shape the activity of mEC SCs by affecting spike repolarization, after‐potentials, excitability and spike patterns. GTx is a useful tool and may serve to further clarify Kv2 channel functions in neurons. We conclude that Kv2 channels in mEC SCs are important determinants of intrinsic properties that allow these neurons to produce spatial representation. The results of the present study may also be important for the accurate modelling of grid cells.

Key points
Both uncoupling protein 1 (UCP1) and UCP3 are important for mammalian thermoregulation.
UCP1 and UCP3 in brown adipose tissue mediate early and late phases of sympathomimetic thermogenesis, respectively.
Lipopolysaccharide thermogenesis requires skeletal muscle UCP3 but not UCP1.
Acute noradrenaline‐induced hyperthermia requires UCP1 but not UCP3.
Loss of both UCP1 and UCP3 accelerate the loss of body temperature compared to UCP1KO alone during acute cold exposure.
Abstract
Uncoupling protein 1 (UCP1) is the established mediator of brown adipose tissue‐dependent thermogenesis. In contrast, the role of UCP3, expressed in both skeletal muscle and brown adipose tissue, in thermoregulatory physiology is less well understood. Here, we show that mice lacking UCP3 (UCP3KO) have impaired sympathomimetic (methamphetamine) and completely abrogated lipopolysaccharide (LPS) thermogenesis, but a normal response to noradrenaline. By comparison, UCP1 knockout (UCP1KO) mice exhibit blunted methamphetamine and fully inhibited noradrenaline thermogenesis, but an increased febrile response to LPS. We further establish that mice lacking both UCP1 and 3 (UCPDK) fail to show methamphetamine‐induced hyperthermia, and have a markedly accelerated loss of body temperature and survival after cold exposure compared to UCP1KO mice. Finally, we show that skeletal muscle‐specific human UCP3 expression is able to significantly rescue LPS, but not sympathomimetic thermogenesis blunted in UCP3KO mice. These studies identify UCP3 as an important mediator of physiological thermogenesis and support a renewed focus on targeting UCP3 in metabolic physiology.

Abstract
This paper is the outcome of the fourth UC Davis Systems Approach to Understanding Cardiac Excitation–Contraction Coupling and Arrhythmias Symposium, a biannual event that aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The theme of the 2016 symposium was ‘K+ Channels and Regulation’. Experts in the field contributed their experimental and mathematical modelling perspectives and discussed emerging questions, controversies and challenges on the topic of cardiac K+ channels. This paper summarizes the topics of formal presentations and informal discussions from the symposium on the structural basis of voltage‐gated K+ channel function, as well as the mechanisms involved in regulation of K+ channel gating, expression and membrane localization. Given the critical role for K+ channels in determining the rate of cardiac repolarization, it is hardly surprising that essentially every aspect of K+ channel function is exquisitely regulated in cardiac myocytes. This regulation is complex and highly interrelated to other aspects of myocardial function. K+ channel regulatory mechanisms alter, and are altered by, physiological challenges, pathophysiological conditions, and pharmacological agents. An accompanying paper focuses on the integrative role of K+ channels in cardiac electrophysiology, i.e. how K+ currents shape the cardiac action potential, and how their dysfunction can lead to arrhythmias, and discusses K+ channel‐based therapeutics. A fundamental understanding of K+ channel regulatory mechanisms and disease processes is fundamental to reveal new targets for human therapy.

Key points
Intracellular pH regulation is vital to neurons as nerve activity produces large and rapid acid loads in presynaptic terminals.
Rapid clearance of acid loads is necessary to maintain control of neurotransmission, but neuronal acid clearance mechanisms remain poorly understood.
Glutamate is loaded into synaptic vesicles via the vesicular glutamate transporter (VGLUT), a mechanism conserved across phyla, and this study reports a previously unknown role for VGLUT as an acid‐extruding protein when deposited in the plasmamembrane during exocytosis.
The finding was made in Drosophila (fruit fly) larval motor neurons through a combined pharamacological and genetic dissection of presynaptic pH homeostatic mechanisms.
A dual role for VGLUT serves to integrate neuronal activity and pH regulation in presynaptic nerve terminals.
Abstract
Neuronal activity can result in transient acidification of presynaptic terminals, and such shifts in cytosolic pH (pHcyto) probably influence mechanisms underlying forms of synaptic plasticity with a presynaptic locus. As neuronal activity drives acid loading in presynaptic terminals, we hypothesized that the same activity might drive acid efflux mechanisms to maintain pHcyto homeostasis. To better understand the integration of neuronal activity and pHcyto regulation we investigated the acid extrusion mechanisms at Drosophila glutamatergic motorneuron terminals. Expression of a fluorescent genetically encoded pH indicator, named ‘pHerry’, in the presynaptic cytosol revealed acid efflux following nerve activity to be greater than that predicted from measurements of the intrinsic rate of acid efflux. Analysis of activity‐induced acid transients in terminals deficient in either endocytosis or exocytosis revealed an acid efflux mechanism reliant upon synaptic vesicle exocytosis. Pharmacological and genetic dissection in situ and in a heterologous expression system indicate that this acid efflux is mediated by conventional plasmamembrane acid transporters, and also by previously unrecognized intrinsic H+/Na+ exchange via the Drosophila vesicular glutamate transporter (DVGLUT). DVGLUT functions not only as a vesicular glutamate transporter but also serves as an acid‐extruding protein when deposited on the plasmamembrane.

Key points
Reaching movements can be perturbed by vestibular input, but the function of this response is unclear.
Here, we applied galvanic vestibular stimulation concurrently with real body movement while subjects maintained arm position either fixed in space or fixed with respect to their body.
During the fixed‐in‐space conditions, galvanic vestibular stimulation caused large changes in arm trajectory consistent with a compensatory response to maintain upper‐limb accuracy in the face of body movement.
Galvanic vestibular stimulation responses were absent during the body‐fixed task, demonstrating task dependency in vestibular control of the upper limb.
The results suggest that the function of vestibular‐evoked arm movements is to maintain the accuracy of the upper limb during unpredictable body movement, but only when reaching in an earth‐fixed reference frame.
Abstract
When using our arms to interact with the world, unintended body motion can introduce movement error. A mechanism that could detect and compensate for such motion would be beneficial. Observations of arm movements evoked by vestibular stimulation provide some support for this mechanism. However, the physiological function underlying these artificially evoked movements is unclear from previous research. For such a mechanism to be functional, it should operate only when the arm is being controlled in an earth‐fixed rather than a body‐fixed reference frame. In the latter case, compensation would be unnecessary and even deleterious. To test this hypothesis, subjects were gently rotated in a chair while being asked to maintain their outstretched arm pointing towards either earth‐fixed or body‐fixed memorized targets. Galvanic vestibular stimulation was applied concurrently during rotation to isolate the influence of vestibular input, uncontaminated by inertial factors. During the earth‐fixed task, galvanic vestibular stimulation produced large polarity‐dependent corrections in arm position. These corrections mimicked those evoked when chair velocity was altered without any galvanic vestibular stimulation, indicating a compensatory arm response to a sensation of altered body motion. In stark contrast, corrections were completely absent during the body‐fixed task, despite the same chair movement profile and arm posture. These effects persisted when we controlled for differences in limb kinematics between the two tasks. Our results demonstrate that vestibular control of the upper limb maintains reaching accuracy during unpredictable body motion. The observation that such responses occurred only when reaching within an earth‐fixed reference frame confirms the functional nature of vestibular‐evoked arm movement.

Key points
In human skeletal muscles, the current view is that the capacity for mitochondrial energy production, and thus endurance capacity, is set by the mitochondria volume.
However, increasing the mitochondrial inner membrane surface comprises an alternative mechanism for increasing the energy production capacity.
In the present study, we show that mitochondrial inner membranes in leg muscles of endurance‐trained athletes have an increased ratio of surface per mitochondrial volume.
We show a positive correlation between this ratio and whole body oxygen uptake and muscle fibre mitochondrial content.
The results obtained in the present study help us to understand modulation of mitochondrial function, as well as how mitochondria can increase their oxidative capacity with increased demand.
Abstract
Mitochondrial energy production involves the movement of protons down a large electrochemical gradient via ATP synthase located on the folded inner membrane, known as cristae. In mammalian skeletal muscle, the density of cristae in mitochondria is assumed to be constant. However, recent experimental studies have shown that respiration per mitochondria varies. Modelling studies have hypothesized that this variation in respiration per mitochondria depends on plasticity in cristae density, although current evidence for such a mechanism is lacking. In the present study, we confirm this hypothesis by showing that, in human skeletal muscle, and in contrast to the current view, the mitochondrial cristae density is not constant but, instead, exhibits plasticity with long‐term endurance training. Furthermore, we show that frequently recruited mitochondria‐enriched fibres have significantly increased cristae density and that, at the whole‐body level, muscle mitochondrial cristae density is a better predictor of maximal oxygen uptake rate than muscle mitochondrial volume. Our findings establish an elevating mitochondrial cristae density as a regulatory mechanism for increasing metabolic power in human skeletal muscle. We propose that this mechanism allows evasion of the trade‐off between cell occupancy by mitochondria and other cellular constituents, as well as improved metabolic capacity and fuel catabolism during prolonged elevated energy requirements.

Key points
Cold water immersion and active recovery are common post‐exercise recovery treatments. A key assumption about the benefits of cold water immersion is that it reduces inflammation in skeletal muscle. However, no data are available from humans to support this notion.
We compared the effects of cold water immersion and active recovery on inflammatory and cellular stress responses in skeletal muscle from exercise‐trained men 2, 24 and 48 h during recovery after acute resistance exercise.
Exercise led to the infiltration of inflammatory cells, with increased mRNA expression of pro‐inflammatory cytokines and neurotrophins, and the subcellular translocation of heat shock proteins in muscle. These responses did not differ significantly between cold water immersion and active recovery.
Our results suggest that cold water immersion is no more effective than active recovery for minimizing the inflammatory and stress responses in muscle after resistance exercise.
Abstract
Cold water immersion and active recovery are common post‐exercise recovery treatments. However, little is known about whether these treatments influence inflammation and cellular stress in human skeletal muscle after exercise. We compared the effects of cold water immersion versus active recovery on inflammatory cells, pro‐inflammatory cytokines, neurotrophins and heat shock proteins (HSPs) in skeletal muscle after intense resistance exercise. Nine active men performed unilateral lower‐body resistance exercise on separate days, at least 1 week apart. On one day, they immersed their lower body in cold water (10°C) for 10 min after exercise. On the other day, they cycled at a low intensity for 10 min after exercise. Muscle biopsies were collected from the exercised leg before, 2, 24 and 48 h after exercise in both trials. Exercise increased intramuscular neutrophil and macrophage counts, MAC1 and CD163 mRNA expression (P

Key points
The lateral habenula (LHb) has been implicated in regulation of drug‐seeking behaviours through aversion‐mediated learning.
In this study, we recorded neuronal activity in the LHb of rats during an operant task before and after ethanol‐induced conditioned taste aversion (CTA) to saccharin.
Ethanol‐induced CTA caused significantly higher baseline firing rates in LHb neurons, as well as elevated firing rates in response to cue presentation, lever press and saccharin taste.
In a separate cohort of rats, we found that bilateral LHb lesions blocked ethanol‐induced CTA.
Our results strongly suggest that excitation of LHb neurons is required for ethanol‐induced CTA, and point towards a mechanism through which LHb firing may regulate voluntary ethanol consumption.
Abstract
Ethanol, like other drugs of abuse, has both rewarding and aversive properties. Previous work suggests that sensitivity to ethanol's aversive effects negatively modulates voluntary alcohol intake and thus may be important in vulnerability to developing alcohol use disorders. We previously found that rats with lesions of the lateral habenula (LHb), which is implicated in aversion‐mediated learning, show accelerated escalation of voluntary ethanol consumption. To understand neural encoding in the LHb contributing to ethanol‐induced aversion, we recorded neural firing in the LHb of freely behaving, water‐deprived rats before and after an ethanol‐induced (1.5 g kg−1 20% ethanol, i.p.) conditioned taste aversion (CTA) to saccharin taste. Ethanol‐induced CTA strongly decreased motivation for saccharin in an operant task to obtain the tastant. Comparison of LHb neural firing before and after CTA induction revealed four main differences in firing properties. First, baseline firing after CTA induction was significantly higher. Second, firing evoked by cues signalling saccharin availability shifted from a pattern of primarily inhibition before CTA to primarily excitation after CTA induction. Third, CTA induction reduced the magnitude of lever press‐evoked inhibition. Finally, firing rates were significantly higher during consumption of the devalued saccharin solution after CTA induction. Next, we studied sham‐ and LHb‐lesioned rats in our operant CTA paradigm and found that LHb lesion significantly attenuated CTA effects in the operant task. Our data demonstrate the importance of LHb excitation in regulating expression of ethanol‐induced aversion and suggest a mechanism for its role in modulating escalation of voluntary ethanol intake.

Key points
Applications of transcranial direct current stimulation to modulate human neuroplasticity have increased in research and clinical settings.
However, the need for longer‐lasting effects, combined with marked inter‐individual variability, necessitates a deeper understanding of the relationship between stimulation parameters and physiological effects.
We systematically investigated the full DC intensity range (0.5–2.0 mA) for both anodal and cathodal tDCS in a sham‐controlled repeated measures design, monitoring changes in motor‐cortical excitability via transcranial magnetic stimulation up to 2 h after stimulation.
For both tDCS polarities, the excitability after‐effects did not linearly correlate with increasing DC intensity; effects of lower intensities (0.5, 1.0 mA) showed equal, if not greater effects in motor‐cortical excitability.
Further, while intra‐individual responses showed good reliability, inter‐individual sensitivity to TMS accounted for a modest percentage of the variance in the early after‐effects of 1.0 mA anodal tDCS, which may be of practical relevance for future optimizations.
Abstract
Contemporary non‐invasive neuromodulatory techniques, such as transcranial direct current stimulation (tDCS), have shown promising potential in both restituting impairments in cortical physiology in clinical settings, as well as modulating cognitive abilities in the healthy population. However, neuroplastic after‐effects of tDCS are highly dependent on stimulation parameters, relatively short lasting, and not expectedly uniform between individuals. The present study systematically investigates the full range of current intensity between 0.5 and 2.0 mA on left primary motor cortex (M1) plasticity, as well as the impact of individual‐level covariates on explaining inter‐individual variability. Thirty‐eight healthy subjects were divided into groups of anodal and cathodal tDCS. Five DC intensities (sham, 0.5, 1.0, 1.5 and 2.0 mA) were investigated in separate sessions. Using transcranial magnetic stimulation (TMS), 25 motor‐evoked potentials (MEPs) were recorded before, and 10 time points up to 2 h following 15 min of tDCS. Repeated‐measures ANOVAs indicated a main effect of intensity for both anodal and cathodal tDCS. With anodal tDCS, all active intensities resulted in equivalent facilitatory effects relative to sham while for cathodal tDCS, only 1.0 mA resulted in sustained excitability diminution. An additional experiment conducted to assess intra‐individual variability revealed generally good reliability of 1.0 mA anodal tDCS (ICC(2,1) = 0.74 over the first 30 min). A post hoc analysis to discern sources of inter‐individual variability confirmed a previous finding in which individual TMS SI1mV (stimulus intensity for 1 mV MEP amplitude) sensitivity correlated negatively with 1.0 mA anodal tDCS effects on excitability. Our study thus provides further insights on the extent of non‐linear intensity‐dependent neuroplastic after‐effects of anodal and cathodal tDCS.

Key points
Glycogen is stored in local spatially distinct compartments within skeletal muscle fibres and is the main energy source during supramaximal exercise.
Using quantitative electron microscopy, we show that supramaximal exercise induces a differential depletion of glycogen from these compartments and also demonstrate how this varies with fibre types.
Repeated exercise alters this compartmentalized glycogen depletion.
The results obtained in the present study help us understand the muscle metabolic dynamics of whole body repeated supramaximal exercise, and suggest that the muscle has a compartmentalized local adaptation to repeated exercise, which affects glycogen depletion.
Abstract
Skeletal muscle glycogen is heterogeneously distributed in three separated compartments (intramyofibrillar, intermyofibrillar and subsarcolemmal). Although only constituting 3–13% of the total glycogen volume, the availability of intramyofibrillar glycogen is of particular importance to muscle function. The present study aimed to investigate the depletion of these three subcellular glycogen compartments during repeated supramaximal exercise in elite athletes. Ten elite cross‐country skiers (aged 25 ± 4 years, V̇O2 max : 65 ± 4 ml kg−1 min−1; mean ± SD) performed four ∼4 min supramaximal sprint time trials (STT 1–4) with 45 min of recovery. The subcellular glycogen volumes in musculus triceps brachii were quantified from electron microscopy images before and after both STT 1 and 4. During STT 1, the depletion of intramyofibrillar glycogen was higher in type 1 fibres [−52%; (−89:−15%)] than type 2 fibres [−15% (−52:22%)] (P = 0.02), whereas the depletion of intermyofibrillar glycogen [main effect: −19% (−33:0%), P = 0.006] and subsarcolemmal glycogen [main effect: −35% (−66:0%), P = 0.03] was similar between fibre types. By contrast, only intermyofibrillar glycogen volume was significantly reduced during STT 4, in both fibre types [main effect: −31% (−50:−11%), P = 0.002]. Furthermore, for each of the subcellular compartments, the depletion of glycogen during STT 1 was associated with the volumes of glycogen before STT 1. In conclusion, the depletion of spatially distinct glycogen compartments differs during supramaximal exercise. Furthermore, the depletion changes with repeated exercise and is fibre type‐dependent.

Key points
Quadrupeds express different gaits depending on speed of locomotion.
Central pattern generators (one per limb) within the spinal cord generate locomotor oscillations and control limb movements. Neural interactions between these generators define interlimb coordination and gait.
We present a computational model of spinal circuits representing four rhythm generators with left–right excitatory and inhibitory commissural and fore–hind inhibitory interactions within the cord.
Increasing brainstem drive to all rhythm generators and excitatory commissural interneurons induces an increasing frequency of locomotor oscillations accompanied by speed‐dependent gait changes from walk to trot and to gallop and bound.
The model closely reproduces and suggests explanations for multiple experimental data, including speed‐dependent gait transitions in intact mice and changes in gait expression in mutants lacking certain types of commissural interneurons. The model suggests the possible circuit organization in the spinal cord and proposes predictions that can be tested experimentally.
Abstract
As speed of locomotion is increasing, most quadrupeds, including mice, demonstrate sequential gait transitions from walk to trot and to gallop and bound. The neural mechanisms underlying these transitions are poorly understood. We propose that the speed‐dependent expression of different gaits results from speed‐dependent changes in the interactions between spinal circuits controlling different limbs and interlimb coordination. As a result, the expression of each gait depends on (1) left–right interactions within the spinal cord mediated by different commissural interneurons (CINs), (2) fore–hind interactions on each side of the spinal cord and (3) brainstem drives to rhythm‐generating circuits and CIN pathways. We developed a computational model of spinal circuits consisting of four rhythm generators (RGs) with bilateral left–right interactions mediated by V0 CINs (V0D and V0V sub‐types) providing left–right alternation, and conditional V3 CINs promoting left–right synchronization. Fore and hind RGs mutually inhibited each other. We demonstrate that linearly increasing excitatory drives to the RGs and V3 CINs can produce a progressive increase in the locomotor speed accompanied by sequential changes of gaits from walk to trot and to gallop and bound. The model closely reproduces and suggests explanations for the speed‐dependent gait expression observed in vivo in intact mice and in mutants lacking V0V or all V0 CINs. Specifically, trot is not expressed after removal of V0V CINs, and only bound is expressed after removal of all V0 CINs. The model provides important insights into the organization of spinal circuits and neural control of locomotion.

Key points
Long‐chain acyl‐CoA synthetase 6 (ACSL6) mRNA is present in human and rat skeletal muscle, and is modulated by nutritional status: exercise and fasting decrease ACSL6 mRNA, whereas acute lipid ingestion increase its expression.
ACSL6 genic inhibition in rat primary myotubes decreased lipid accumulation, as well as activated the higher mitochondrial oxidative capacity programme and fatty acid oxidation through the AMPK/PGC1‐α pathway.
ACSL6 overexpression in human primary myotubes increased phospholipid species and decreased oxidative metabolism.
Abstract
Long‐chain acyl‐CoA synthetases (ACSL 1 to 6) are key enzymes regulating the partitioning of acyl‐CoA species toward different metabolic fates such as lipid synthesis or β‐oxidation. Despite our understanding of ecotopic lipid accumulation in skeletal muscle being associated with metabolic diseases such as obesity and type II diabetes, the role of specific ACSL isoforms in lipid synthesis remains unclear. In the present study, we describe for the first time the presence of ACSL6 mRNA in human skeletal muscle and the role that ACSL6 plays in lipid synthesis in both rodent and human skeletal muscle. ACSL6 mRNA was observed to be up‐regulated by acute high‐fat meal ingestion in both rodents and humans. In rats, we also demonstrated that fasting and chronic aerobic training negatively modulated the ACSL6 mRNA and other genes of lipid synthesis. Similar results were obtained following ACSL6 knockdown in rat myotubes, which was associated with a decreased accumulation of TAGs and lipid droplets. Under the same knockdown condition, we further demonstrate an increase in fatty acid content, p‐AMPK, mitochondrial content, mitochondrial respiratory rates and palmitate oxidation. These results were associated with increased PGC‐1α, UCP2 and UCP3 mRNA and decreased reactive oxygen species production. In human myotubes, ACSL6 overexpression reduced palmitate oxidation and PGC‐1α mRNA. In conclusion, ACSL6 drives acyl‐CoA toward lipid synthesis and its downregulation improves mitochondrial biogenesis, respiratory capacity and lipid oxidation. These outcomes are associated with the activation of the AMPK/PGC1‐α pathway.

Key points
Phosphatidylinositol‐4,5‐bisphosphate (PIP2) is a key regulator of many membrane proteins, including voltage‐gated Kv7.2 channels.
In this study, we identified the residues in five phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP2‐binding domains in Kv7.2.
Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP2.
Dephosphorylation of Kv7.2 affected channel inhibition via M1 muscarinic receptors, but not via bradykinin receptors.
Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP2, thereby ensuring the tight regulation of the channel via G protein‐coupled receptors.
Abstract
The function of numerous ion channels is tightly controlled by G protein‐coupled receptors (GPCRs). The underlying signalling mechanisms may involve phosphorylation of channel proteins and participation of phosphatidylinositol‐4,5‐bisphosphate (PIP2). Although the roles of both mechanisms have been investigated extensively, thus far only little has been reported on their interaction in channel modulation. GPCRs govern Kv7 channels, the latter playing a major role in the regulation of neuronal excitability by determining the levels of PIP2 and through phosphorylation. Using liquid chromatography‐coupled mass spectrometry for Kv7.2 immunoprecipitates of rat brain membranes and transfected cells, we mapped a cluster of five phosphorylation sites in one of the PIP2‐binding domains. To evaluate the effect of phosphorylation on PIP2‐mediated Kv7.2 channel regulation, a quintuple alanine mutant of these serines (S427/S436/S438/S446/S455; A5 mutant) was generated to mimic the dephosphorylated state. Currents passing through these mutated channels were less sensitive towards PIP2 depletion via the voltage‐sensitive phosphatase Dr‐VSP than were wild‐type channels. In vitro phosphorylation assays with the purified C‐terminus of Kv7.2 revealed that CDK5, p38 MAPK, CaMKIIα and PKA were able to phosphorylate the five serines. Inhibition of these protein kinases reduced the sensitivity of wild‐type but not mutant Kv7.2 channels towards PIP2 depletion via Dr‐VSP. In superior cervical ganglion neurons, the protein kinase inhibitors attenuated Kv7 current regulation via M1 receptors, but left unaltered the control by B2 receptors. Our results revealed that the phosphorylation status of serines located within a putative PIP2‐binding domain determined the phospholipid sensitivity of Kv7.2 channels and supported GPCR‐mediated channel regulation.

Key points
There have been few systematic population‐wide analyses of relationships between spike synchrony within a period of several milliseconds and behavioural functions.
In this study, we obtained a large amount of spike data from > 23,000 neuron pairs by multiple single‐unit recording from deep layer neurons in motor cortical areas in rats performing a forelimb movement task.
The temporal changes of spike synchrony in the whole neuron pairs were statistically independent of behavioural changes during the task performance, although some neuron pairs exhibited correlated changes in spike synchrony.
Mutual information analyses revealed that spike synchrony made a smaller contribution than spike rate to behavioural functions.
The strength of spike synchrony between two neurons was statistically independent of the spike rate‐based preferences of the pair for behavioural functions.
Abstract
Spike synchrony within a period of several milliseconds in presynaptic neurons enables effective integration of functional information in the postsynaptic neuron. However, few studies have systematically analysed the population‐wide relationships between spike synchrony and behavioural functions. Here we obtained a sufficiently large amount of spike data among regular‐spiking (putatively excitatory) and fast‐spiking (putatively inhibitory) neuron subtypes (> 23,000 pairs) by multiple single‐unit recording from deep layers in motor cortical areas (caudal forelimb area, rostral forelimb area) in rats performing a forelimb movement task. After holding a lever, rats pulled the lever either in response to a cue tone (external‐trigger trials) or spontaneously without any cue (internal‐trigger trials). Many neurons exhibited functional spike activity in association with forelimb movements, and the preference of regular‐spiking neurons in the rostral forelimb area was more biased toward externally triggered movement than that in the caudal forelimb area. We found that a population of neuron pairs with spike synchrony does exist, and that some neuron pairs exhibit a dependence on movement phase during task performance. However, the population‐wide analysis revealed that spike synchrony was statistically independent of the movement phase and the spike rate‐based preferences of the pair for behavioural functions, whereas spike rates were clearly dependent on the movement phase. In fact, mutual information analyses revealed that the contribution of spike synchrony to the behavioural functions was small relative to the contribution of spike rate. Our large‐scale analysis revealed that cortical spike rate, rather than spike synchrony, contributes to population coding for movement.

Key points
A polymorphism of human AE3 is associated with idiopathic generalized epilepsy. Knockout of AE3 in mice lowers the threshold for triggering epileptic seizures. The explanations for these effects are elusive.
Comparisons of cells from wild‐type vs. AE3–/– mice show that AE3 (present in hippocampal neurons, not astrocytes; mediates HCO3– efflux) enhances intracellular pH (pHi) recovery (decrease) from alkali loads in neurons and, surprisingly, adjacent astrocytes.
During metabolic acidosis (MAc), AE3 speeds initial acidification, but limits the extent of pHi decrease in neurons and astrocytes.
AE3 speeds re‐alkalization after removal of MAc in neurons and astrocytes, and speeds neuronal pHi recovery from an ammonium prepulse‐induced acid load.
We propose that neuronal AE3 indirectly increases acid extrusion in (a) neurons via Cl– loading, and (b) astrocytes by somehow enhancing NBCe1 (major acid extruder). The latter would enhance depolarization‐induced alkalinization of astrocytes, and extracellular acidification, and thereby reduce susceptibility to epileptic seizures.
Abstract
The anion exchanger AE3, expressed in hippocampal (HC) neurons but not astrocytes, contributes to intracellular pH (pHi) regulation by facilitating the exchange of extracellular Cl– for intracellular HCO3–. The human AE3 polymorphism A867D is associated with idiopathic generalized epilepsy. Moreover, AE3 knockout (AE3–/–) mice are more susceptible to epileptic seizure. The mechanism of these effects has been unclear because the starting pHi in AE3–/– and wild‐type neurons is indistinguishable. The purpose of the present study was to use AE3–/– mice to investigate the role of AE3 in pHi homeostasis in HC neurons, co‐cultured with astrocytes. We find that the presence of AE3 increases the acidification rate constant during pHi recovery from intracellular alkaline loads imposed by reducing [CO2]. The presence of AE3 also speeds intracellular acidification during the early phase of metabolic acidosis (MAc), not just in neurons but, surprisingly, in adjacent astrocytes. Additionally, AE3 contributes to braking the decrease in pHi later during MAc in both neurons and astrocytes. Paradoxically, AE3 enhances intracellular re‐alkalization after MAc removal in neurons and astrocytes, and pHi recovery from an ammonium prepulse‐induced acid load in neurons. The effects of AE3 knockout on astrocytic pHi homeostasis in MAc‐related assays require the presence of neurons, and are consistent with the hypothesis that the AE3 knockout reduces functional expression of astrocytic NBCe1. These findings suggest a new type of neuron–astrocyte communication, based on the expression of AE3 in neurons, which could explain how AE3 reduces seizure susceptibility.

Key points
In the synaptic cleft between type I hair cells and calyceal afferents, K+ ions accumulate as a function of activity, dynamically altering the driving force and permeation through ion channels facing the synaptic cleft.
High‐fidelity synaptic transmission is possible due to large conductances that minimize hair cell and afferent time constants in the presence of significant membrane capacitance.
Elevated potassium maintains hair cells near a potential where transduction currents are sufficient to depolarize them to voltages necessary for calcium influx and synaptic vesicle fusion.
Elevated potassium depolarizes the postsynaptic afferent by altering ion permeation through hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels, and contributes to depolarizing the afferent to potentials where a single EPSP (quantum) can generate an action potential.
With increased stimulation, hair cell depolarization increases the frequency of quanta released, elevates [K+]cleft and depolarizes the afferent to potentials at which smaller and smaller EPSPs would be sufficient to trigger APs.
Abstract
Fast neurotransmitters act in conjunction with slower modulatory effectors that accumulate in restricted synaptic spaces found at giant synapses such as the calyceal endings in the auditory and vestibular systems. Here, we used dual patch‐clamp recordings from turtle vestibular hair cells and their afferent neurons to show that potassium ions accumulating in the synaptic cleft modulated membrane potentials and extended the range of information transfer. High‐fidelity synaptic transmission was possible due to large conductances that minimized hair cell and afferent time constants in the presence of significant membrane capacitance. Increased potassium concentration in the cleft maintained the hair cell near potentials that promoted the influx of calcium necessary for synaptic vesicle fusion. The elevated potassium concentration also depolarized the postsynaptic neuron by altering ion permeation through hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels. This depolarization enabled the afferent to reliably generate action potentials evoked by single AMPA‐dependent EPSPs. Depolarization of the postsynaptic afferent could also elevate potassium in the synaptic cleft, and would depolarize other hair cells enveloped by the same neuritic process increasing the fidelity of neurotransmission at those synapses as well. Collectively, these data demonstrate that neuronal activity gives rise to potassium accumulation, and suggest that potassium ion action on HCN channels can modulate neurotransmission, preserving the fidelity of high‐speed synaptic transmission by dynamically shifting the resting potentials of both presynaptic and postsynaptic cells.

Key points
Marijuana was found to cause muscle weakness, although the exact regulatory role of its receptors (CB1 cannabinoid receptor; CB1R) in the excitation–contraction coupling (ECC) of mammalian skeletal muscle remains unknown.
We found that CB1R activation or its knockout did not affect muscle force directly, whereas its activation decreased the Ca2+‐sensitivity of the contractile apparatus and made the muscle fibres more prone to fatigue.
We demonstrate that CB1Rs are not connected to the inositol 1,4,5‐trisphosphate pathway either in myotubes or in adult muscle fibres.
By contrast, CB1Rs constitutively inhibit sarcoplasmic Ca2+ release and sarcoplasmic reticulum Ca2+ ATPase during ECC in a Gi/o protein‐mediated way in adult skeletal muscle fibres but not in myotubes.
These results help with our understanding of the physiological effects and pathological consequences of CB1R activation in skeletal muscle and may be useful in the development of new cannabinoid drugs.
Abstract
Marijuana was found to cause muscle weakness, although it is unknown whether it affects the muscles directly or modulates only the motor control of the central nervous system. Although the presence of CB1 cannabinoid receptors (CB1R), which are responsible for the psychoactive effects of the drug in the brain, have recently been demonstrated in skeletal muscle, it is unclear how CB1R‐mediated signalling affects the contraction and Ca²⁺ homeostasis of mammalian skeletal muscle. In the present study, we demonstrate that in vitro CB1R activation increased muscle fatigability and decreased the Ca2+‐sensitivity of the contractile apparatus, whereas it did not alter the amplitude of single twitch contractions. In myotubes, CB1R agonists neither evoked, nor influenced inositol 1,4,5‐trisphosphate (IP3)‐mediated Ca2+ transients, nor did they alter excitation–contraction coupling. By contrast, in isolated muscle fibres of wild‐type mice, although CB1R agonists did not evoke IP3‐mediated Ca2+ transients too, they significantly reduced the amplitude of the depolarization‐evoked transients in a pertussis‐toxin sensitive manner, indicating a Gi/o protein‐dependent mechanism. Concurrently, on skeletal muscle fibres isolated from CB1R‐knockout animals, depolarization‐evoked Ca2+ transients, as well qas Ca2+ release flux via ryanodine receptors (RyRs), and the total amount of released Ca2+ was significantly greater than that from wild‐type mice. Our results show that CB1R‐mediated signalling exerts both a constitutive and an agonist‐mediated inhibition on the Ca2+ transients via RyR, regulates the activity of the sarcoplasmic reticulum Ca2+ ATPase and enhances muscle fatigability, which might decrease exercise performance, thus playing a role in myopathies, and therefore should be considered during the development of new cannabinoid drugs.

Key points
It is recognized that seizures commonly cause apnoea and oxygen desaturation, but there is still a lack in the literature about the respiratory impairments observed ictally and in the post‐ictal period.
Respiratory disorders may involve changes in serotonergic transmission at the level of the retrotrapezoid nucleus (RTN).
In this study, we evaluated breathing activity and the role of serotonergic transmission in the RTN with a rat model of tonic–clonic seizures, the Wistar audiogenic rat (WAR).
We conclude that the respiratory impairment in the WAR could be correlated to an overall decrease in the number of neurons located in the respiratory column.
Abstract
Respiratory disorders may involve changes in serotonergic neurotransmission at the level of the chemosensitive neurons located in the retrotrapezoid nucleus (RTN). Here, we investigated the central respiratory chemoreflex and the role of serotonergic neurotransmission in the RTN with a rat model of tonic–clonic seizures, the Wistar audiogenic rat (WAR). We found that naive or kindled WARs have reduced resting ventilation and ventilatory response to hypercapnia (7% CO2). The number of chemically coded (Phox2b+/TH−) RTN neurons, as well as the serotonergic innervation to the RTN, was reduced in WARs. We detected that the ventilatory response to serotonin (1 mm, 50 nl) within the RTN region was significantly reduced in WARs. Our results uniquely demonstrated a respiratory impairment in a genetic model of tonic–clonic seizures, the WAR strain. More importantly, we demonstrated an overall decrease in the number of neurons located in the ventral respiratory column (VRC), as well as a reduction in serotonergic neurons in the midline medulla. This is an important step forward to demonstrate marked changes in neuronal activity and breathing impairment in the WAR strain, a genetic model of epilepsy.

Key points
Emotions are accompanied by concordant changes in visceral function, including cardiac output, respiration and digestion. One major forebrain integrator of emotional responses, the amygdala, is considered to rely on embedded visceral afferent information, although few details are known.
In the present study, we retrogradely transported dye from the central nucleus of the amygdala (CeA) to identify CeA‐projecting nucleus of the solitary tract (NTS) neurons for synaptic characterization and compared them with unlabelled, near‐neighboor NTS neurons.
Solitary tract (ST) afferents converged onto NTS‐CeA second‐order sensory neurons in greater numbers, as well as indirectly via polysynaptic pathways.
Unexpectedly, all mono‐ and polysynaptic ST afferent pathways to NTS‐CeA neurons were organized exclusively as either transient receptor potential cation channel subfamily V member 1 (TRPV1)‐sensitive or TRPV1‐resistant, regardless of whether intervening neurons were excitatory or inhibitory.
This strict sorting provides viscerosensory signals to CeA about visceral conditions with respect to being either ‘normal’ via A‐fibres or ‘alarm’ via TRPV1 expressing C‐fibres and, accordingly, this pathway organization probably encodes interoceptive status.
Abstract
Emotional state is impacted by changes in visceral function, including blood pressure, breathing and digestion. A main line of viscerosensory information processing occurs first in the nucleus of the solitary tract (NTS). In the present study conducted in rats, we examined the synaptic characteristics of visceral afferent pathways to the central nucleus of the amygdala (CeA) in brainstem slices by recording from retrogradely labelled NTS projection neurons. We simultaneously recorded neuron pairs: one dye positive (i.e. NTS‐CeA) and a second unlabelled neighbour. Graded shocks to the solitary tract (ST) always (93%) triggered EPSCs at CeA projecting NTS neurons. Half of the NTS‐CeA neurons received at least one primary afferent input (classed ‘second order’) indicating that viscerosensory information arrives at the CeA conveyed via a pathway involving as few as two synapses. The remaining NTS‐CeA neurons received viscerosensory input only via polysynaptic pathways. By contrast, ∼3/4 of unlabelled neighbouring neurons were directly connected to ST. NTS‐CeA neurons received greater numbers of ST‐related inputs compared to unlabelled NTS neurons, indicating that highly convergent viscerosensory signals reach the CeA. Remarkably, despite multifibre convergence, all single NTS‐CeA neurons received inputs derived from only unmyelinated afferents [transient receptor potential cation channel subfamily V member 1 (TRPV1) expressing C‐fibres] or only non‐TRPV1 ST afferent inputs, and never a combination of both. Such segregation means that visceral afferent information followed separate lines to reach the CeA. Their very different physiological activation profiles mean that these parallel visceral afferent pathways encode viscerosensory signals to the amygdala that may provide interoceptive assessments to impact on behaviours.

Key points
Mitochondria are frequently implicated in the ageing of skeletal muscle, although the role of denervation in modulating mitochondrial function in ageing muscle is unknown.
We show that increased sensitivity to apoptosis initiation occurs prior to evidence of persistent denervation and is thus a primary mitochondrial defect in ageing muscle worthy of therapeutic targeting.
However, at more advanced age, mitochondrial function changes are markedly impacted by persistent sporadic myofibre denervation, suggesting the mitochondrion may be a less viable therapeutic target.
Abstract
Experimental denervation modulates mitochondrial function, where changes in both reactive oxygen species (ROS) and sensitivity to permeability transition are implicated in the resultant muscle atrophy. Notably, although denervation occurs sporadically in ageing muscle, its impact on ageing muscle mitochondria is unknown. Because this information has important therapeutic implications concerning targeting the mitochondrion in ageing muscle, we examined mitochondrial function in skeletal muscle from four groups of humans, comprising two active (mean ± SD age: 23.7 ± 2.7 years and 71.2 ± 4.9 years) and two inactive groups (64.8 ± 3.1 years and 82.5 ± 4.8 years), and compared this with a murine model of sporadic denervation. We tested the hypothesis that, although some alterations of mitochondrial function in aged muscle are attributable to a primary organelle defect, mitochondrial dysfunction would be impacted by persistent denervation in advanced age. Both ageing in humans and sporadic denervation in mice increased mitochondrial sensitivity to permeability transition (humans, P = 0.004; mice, P = 0.01). To determine the contribution of sporadic denervation to mitochondrial function, we pharmacologically inhibited the denervation‐induced ROS response. This reduced ROS emission by 60% (P = 0.02) in sporadically denervated mouse muscle, which is similar to that seen in humans older than 75 years (–66%, P = 0.02) but not those younger than 75 years. We conclude that an increased sensitivity to permeability transition is a primary mitochondrial defect in ageing muscle. However, at more advanced age, when muscle atrophy becomes more clinically severe, mitochondrial function changes are markedly impacted by persistent sporadic denervation, making the mitochondrion a less viable therapeutic target.

Key points
Sinoatrial node (SAN) function declines with age; however, not all individuals age at the same rate and health status can vary from fit to frail.
Frailty was quantified in young and aged mice using a non‐invasive frailty index so that the impacts of age and frailty on heart rate and SAN function could be assessed.
SAN function was impaired in aged mice due to alterations in electrical conduction, changes in SAN action potential morphology and fibrosis in the SAN.
Changes in SAN function, electrical conduction, action potential morphology and fibrosis were correlated with, and graded by, frailty.
This study shows that mice of the same chronological age have quantifiable differences in health status that impact heart rate and SAN function and that these differences in health status can be identified using our frailty index.
Abstract
Sinoatrial node (SAN) dysfunction increases with age, although not all older adults are affected in the same way. This is because people age at different rates and individuals of the same chronological age vary in health status from very fit to very frail. Our objective was to determine the impacts of age and frailty on heart rate (HR) and SAN function using a new model of frailty in ageing mice. Frailty, which was quantified in young and aged mice using a frailty index (FI), was greater in aged vs. young mice. Intracardiac electrophysiology demonstrated that HR was reduced whereas SAN recovery time (SNRT) was prolonged in aged mice; however, both parameters showed heteroscedasticity suggesting differences in health status among mice of similar chronological age. Consistent with this, HR and corrected SNRT were correlated with, and graded by, FI score. Optical mapping of the SAN demonstrated that conduction velocity (CV) was reduced in aged hearts in association with reductions in diastolic depolarization (DD) slope and action potential (AP) duration. In agreement with in vivo results, SAN CV, DD slope and AP durations all correlated with FI score. Finally, SAN dysfunction in aged mice was associated with increased interstitial fibrosis and alterations in expression of matrix metalloproteinases, which also correlated with frailty. These findings demonstrate that age‐related SAN dysfunction occurs in association with electrical and structural remodelling and that frailty is a critical determinant of health status of similarly aged animals that correlates with changes in HR and SAN function.

Key points
Inhibiting Nox2 reactive oxygen species (ROS) production reduced in vivo calcium influx in dystrophic muscle.
The lack of Nox2 ROS production protected against decreased in vivo muscle function in dystrophic mice.
Manganese‐enhanced magnetic resonance imaging (MEMRI) was able to detect alterations in basal calcium levels in skeletal muscle and differentiate disease status.
Administration of Mn2+ did not affect muscle function or the health of the animal, and Mn2+ was cleared from skeletal muscle rapidly.
We conclude that MEMRI may be a viable, non‐invasive technique to monitor molecular alterations in disease progression and evaluate the effectiveness of potential therapies for Duchenne muscular dystrophy.
Abstract
Duchenne muscular dystrophy (DMD) is an X‐linked progressive degenerative disease resulting from a mutation in the gene that encodes dystrophin, leading to decreased muscle mechanical stability and force production. Increased Nox2 reactive oxygen species (ROS) production and sarcolemmal Ca2+ influx are early indicators of disease pathology, and eliminating Nox2 ROS production reduces aberrant Ca2+ influx in young mdx mice, a model of DMD. Various imaging modalities have been used to study dystrophic muscle in vivo; however, they are based upon alterations in muscle morphology or inflammation. Manganese has been used for indirect monitoring of calcium influx across the sarcolemma and may allow detection of molecular alterations in disease progression in vivo using manganese‐enhanced magnetic resonance imaging (MEMRI). Therefore, we hypothesized that eliminating Nox2 ROS production would decrease calcium influx in adult mdx mice and that MEMRI would be able to monitor and differentiate disease status in dystrophic muscle. Both in vitro and in vivo data demonstrate that eliminating Nox2 ROS protected against aberrant Ca2+ influx and improved muscle function in dystrophic muscle. MEMRI was able to differentiate between different pathological states in vivo, with no long‐term effects on animal health or muscle function. We conclude that MEMRI is a viable, non‐invasive technique to differentiate disease status and might provide a means to monitor and evaluate the effectiveness of potential therapies in dystrophic muscle.

Key points
Candesartan, an inverse agonist of the type 1 angiotensin II receptor (AT1R), causes a concentration‐dependent inhibition of pressure‐dependent myogenic tone consistent with previous reports of mechanosensitivity of this G protein‐coupled receptor.
Mechanoactivation of the AT1R occurs independently of local angiotensin II production and the type 2 angiotensin receptor.
Mechanoactivation of the AT1R stimulates actin polymerization by a protein kinase C‐dependent mechanism, but independently of a change in intracellular Ca2+.
Using atomic force microscopy, changes in single vascular smooth muscle cell cortical actin are observed to remodel following mechanoactivation of the AT1R.
Abstract
The Gq/11 protein‐coupled angiotensin II type 1 receptor (AT1R) has been shown to be activated by mechanical stimuli. In the vascular system, evidence supports the AT1R being a mechanosensor that contributes to arteriolar myogenic constriction. The aim of this study was to determine if AT1R mechanoactivation affects myogenic constriction in skeletal muscle arterioles and to determine underlying cellular mechanisms. Using pressure myography to study rat isolated first‐order cremaster muscle arterioles the AT1R inhibitor candesartan (10−7–10−5 m) showed partial but concentration‐dependent inhibition of myogenic reactivity. Inhibition was demonstrated by a rightward shift in the pressure–diameter relationship over the intraluminal pressure range, 30–110 mmHg. Pressure‐induced changes in global vascular smooth muscle intracellular Ca2+ (using Fura‐2) were similar in the absence or presence of candesartan, indicating that AT1R‐mediated myogenic constriction relies on Ca2+‐independent downstream signalling. The diacylglycerol analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) reversed the inhibitory effect of candesartan, while this rescue effect was prevented by the protein kinase C (PKC) inhibitor GF 109203X. Both candesartan and PKC inhibition caused increased G‐actin levels, as determined by Western blotting of vessel lysates, supporting involvement of cytoskeletal remodelling. At the single vascular smooth muscle cell level, atomic force microscopy showed that cell swelling (stretch) with hypotonic buffer also caused thickening of cortical actin fibres and this was blocked by candesartan. Collectively, the present studies support growing evidence for novel modes of activation of the AT1R in arterioles and suggest that mechanically activated AT1R generates diacylglycerol, which in turn activates PKC which induces the actin cytoskeleton reorganization that is required for pressure‐induced vasoconstriction.

Key points
Spinal reflexes are substantial components of the motor control system in all vertebrates and centrally driven reflex modifications are essential to many behaviours, but little is known about the neuronal mechanisms underlying these modifications.
To study this issue, we took advantage of an in vitro brainstem–spinal cord preparation of the lamprey (a lower vertebrate), in which spinal reflex responses to spinal cord bending (caused by signals from spinal stretch receptor neurons) can be evoked during different types of fictive behaviour.
Our results demonstrate that reflexes observed during fast forward swimming are reversed during escape behaviours, with the reflex reversal presumably caused by supraspinal commands transmitted by a population of reticulospinal neurons.
NMDA receptors are involved in the formation of these commands, which are addressed primarily to the ipsilateral spinal networks.
In the present study the neuronal mechanisms underlying reflex reversal have been characterized for the first time.
Abstract
Spinal reflexes can be modified during different motor behaviours. However, our knowledge about the neuronal mechanisms underlying these modifications in vertebrates is scarce. In the lamprey, a lower vertebrate, body bending causes activation of intraspinal stretch receptor neurons (SRNs) resulting in spinal reflexes: activation of motoneurons (MNs) with bending towards either the contralateral or ipsilateral side (a convex or concave response, respectively). The present study had two main aims: (i) to investigate how these spinal reflexes are modified during different motor behaviours, and (ii) to reveal reticulospinal neurons (RSNs) transmitting commands for the reflex modification. For this purpose in in vitro brainstem–spinal cord preparation, RSNs and reflex responses to bending were recorded during different fictive behaviours evoked by supraspinal commands. We found that during fast forward swimming MNs exhibited convex responses. By contrast, during escape behaviours, MNs exhibited concave responses. We found RSNs that were activated during both stimulation causing reflex reversal without initiation of any specific behaviour, and stimulation causing reflex reversal during escape behaviour. We suggest that these RSNs transmit commands for the reflex modification. Application of the NMDA antagonist (AP‐5) to the brainstem significantly decreased the reversed reflex, suggesting involvement of NMDA receptors in the formation of these commands. Longitudinal split of the spinal cord did not abolish the reflex reversal caused by supraspinal commands, suggesting an important role for ipsilateral networks in determining this type of motor response. This is the first study to reveal the neuronal mechanisms underlying supraspinal control of reflex reversal.

Key points
Synaptic transmission at the endbulb of Held was assessed by whole‐cell patch clamp recordings from auditory neurons in mature (2–4 months) and aged (20–26 months) mice.
Synaptic transmission is degraded in aged mice, which may contribute to the decline in neural processing of the central auditory system during age‐related hearing loss.
The changes in synaptic transmission in aged mice can be partially rescued by improving calcium buffering, or decreasing action potential‐evoked calcium influx.
These experiments suggest potential mechanisms, such as regulating intraterminal calcium, that could be manipulated to improve the fidelity of transmission at the aged endbulb of Held.
Abstract
Age‐related hearing loss (ARHL) is associated with changes to the auditory periphery that raise sensory thresholds and alter coding, and is accompanied by alterations in excitatory and inhibitory synaptic transmission, and intrinsic excitability in the circuits of the central auditory system. However, it remains unclear how synaptic transmission changes at the first central auditory synapses during ARHL. Using mature (2–4 months) and old (20–26 months) CBA/CaJ mice, we studied synaptic transmission at the endbulb of Held. Mature and old mice showed no difference in either spontaneous quantal synaptic transmission or low frequency evoked synaptic transmission at the endbulb of Held. However, when challenged with sustained high frequency stimulation, synapses in old mice exhibited increased asynchronous transmitter release and reduced synchronous release. This suggests that the transmission of temporally precise information is degraded at the endbulb during ARHL. Increasing intraterminal calcium buffering with EGTA‐AM or decreasing calcium influx with ω‐agatoxin IVA decreased the amount of asynchronous release and restored synchronous release in old mice. In addition, recovery from depression following high frequency trains was faster in old mice, but was restored to a normal time course by EGTA‐AM treatment. These results suggest that intraterminal calcium in old endbulbs may rise to abnormally high levels during high rates of auditory nerve firing, or that calcium‐dependent processes involved in release are altered with age. These observations suggest that ARHL is associated with a decrease in temporal precision of synaptic release at the first central auditory synapse, which may contribute to perceptual deficits in hearing.

Key points
Biotin, a vitamin whose main role is as a coenzyme for carboxylases, accumulates at unusually large amounts within cells of the carotid body (CB).
In biotin‐deficient rats biotin rapidly disappears from the blood; however, it remains at relatively high levels in CB glomus cells. The CB contains high levels of mRNA for SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin.
Animals with biotin deficiency exhibit pronounced metabolic lactic acidosis. Remarkably, glomus cells from these animals have normal electrical and neurochemical properties. However, they show a marked decrease in the size of quantal dopaminergic secretory events.
Inhibitors of the vesicular monoamine transporter 2 (VMAT2) mimic the effect of biotin deficiency. In biotin‐deficient animals, VMAT2 protein expression decreases in parallel with biotin depletion in CB cells.
These data suggest that dopamine transport and/or storage in small secretory granules in glomus cells depend on biotin.
Abstract
Biotin is a water‐soluble vitamin required for the function of carboxylases as well as for the regulation of gene expression. Here, we report that biotin accumulates in unusually large amounts in cells of arterial chemoreceptors, carotid body (CB) and adrenal medulla (AM). We show in a biotin‐deficient rat model that the vitamin rapidly disappears from the blood and other tissues (including the AM), while remaining at relatively high levels in the CB. We have also observed that, in comparison with other peripheral neural tissues, CB cells contain high levels of SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Biotin‐deficient rats show a syndrome characterized by marked weight loss, metabolic lactic acidosis, aciduria and accelerated breathing with normal responsiveness to hypoxia. Remarkably, CB cells from biotin‐deficient animals have normal electrophysiological and neurochemical (ATP levels and catecholamine synthesis) properties; however, they exhibit a marked decrease in the size of quantal catecholaminergic secretory events, which is not seen in AM cells. A similar differential secretory dysfunction is observed in CB cells treated with tetrabenazine, a selective inhibitor of the vesicular monoamine transporter 2 (VMAT2). VMAT2 is highly expressed in glomus cells (in comparison with VMAT1), and in biotin‐deficient animals VMAT2 protein expression decreases in parallel with the decrease of biotin accumulated in CB cells. These data suggest that biotin has an essential role in the homeostasis of dopaminergic transmission modulating the transport and/or storage of transmitters within small secretory granules in glomus cells.

Key points
In uninjured humans, transmission in the corticospinal pathway changes in a task‐dependent manner during movement preparation. We investigated whether this ability is preserved in humans with incomplete chronic cervical spinal cord injury (SCI).
Our results show that corticospinal excitability is altered in the preparatory phase of an upcoming movement when there is a need to suppress but not to execute rapid index finger voluntary contractions in individuals with SCI compared with controls. This is probably related to impaired transmission at a cortical and spinal level after SCI.
Overall our findings indicate that deficits in corticospinal transmission in humans with chronic incomplete SCI are also present in the preparatory phase of upcoming movements.
Abstract
Corticospinal output is modulated in a task‐dependent manner during the preparatory phase of upcoming movements in humans. Whether this ability is preserved after spinal cord injury (SCI) is unknown. In this study, we examined motor evoked potentials elicited by cortical (MEPs) and subcortical (CMEPs) stimulation of corticospinal axons and short‐interval intracortical inhibition in the first dorsal interosseous muscle in the preparatory phase of a reaction time task where individuals with chronic incomplete cervical SCI and age‐matched controls needed to suppress (NOGO) or initiate (GO) ballistic index finger isometric voluntary contractions. Reaction times were prolonged in SCI participants compared with control subjects and stimulation was provided ∼90 ms prior to movement onset in each group. During NOGO trials, both MEPs and CMEPs remained unchanged compared to baseline in SCI participants but were suppressed in control subjects. Notably, during GO trials, MEPs increased to a similar extent in both groups but CMEPs increased only in controls. The magnitude of short‐interval intracortical inhibition increased in controls but not in SCI subjects during NOGO trials and decreased in both groups in GO trials. These novel observations reveal that humans with incomplete cervical SCI have an altered ability to modulate corticospinal excitability during movement preparation when there is a need to suppress but not to execute upcoming rapid finger movements, which is probably related to impaired transmission at a cortical and spinal level. Thus, deficits in corticospinal transmission after human SCI extend to the preparatory phase of upcoming movements.

Key points
Loss of function of the melanocortin 4 receptor (MC4R) results in hyperphagia, obesity and increased growth.
Despite knowing that MC4Rs control food intake, we are yet to understand why defects in the function of the MC4R receptor contribute to rapid linear growth.
We show that hyperphagia following germline loss of MC4R in male mice promotes growth while suppressing the growth hormone–insulin‐like growth factor‐1 (GH–IGF‐1) axis.
We propose that hyperinsulinaemia promotes growth while suppressing the GH–IGF‐1 axis.
It is argued that physiological responses essential to maintain energy flux override conventional mechanisms of pubertal growth to promote the storage of excess energy while ensuring growth.
Abstract
Defects in melanocortin‐4‐receptor (MC4R) signalling result in hyperphagia, obesity and increased growth. Clinical observations suggest that loss of MC4R function may enhance growth hormone (GH)‐mediated growth, although this remains untested. Using male mice with germline loss of the MC4R, we assessed pulsatile GH release and insulin‐like growth factor‐1 (IGF‐1) production and/or release relative to pubertal growth. We demonstrate early‐onset suppression of GH release in rapidly growing MC4R deficient (MC4RKO) mice, confirming that increased linear growth in MC4RKO mice does not occur in response to enhanced activation of the GH–IGF‐1 axis. The progressive suppression of GH release in MC4RKO mice occurred alongside increased adiposity and the progressive worsening of hyperphagia‐associated hyperinsulinaemia. We next prevented hyperphagia in MC4RKO mice through restricting calorie intake in these mice to match that of wild‐type (WT) littermates. Pair feeding of MC4RKO mice did not prevent increased adiposity, but attenuated hyperinsulinaemia, recovered GH release, and normalized linear growth rate to that seen in pair‐fed WT littermate controls. We conclude that the suppression of GH release in MC4RKO mice occurs independently of increased adipose mass, and is a consequence of hyperphagia‐associated hyperinsulinaemia. It is proposed that physiological responses essential to maintain energy flux (hyperinsulinaemia and the suppression of GH release) override conventional mechanisms of pubertal growth to promote the storage of excess energy while ensuring growth. Implications of these findings are likely to extend beyond individuals with defects in MC4R signalling, encompassing physiological changes central to mechanisms of growth and energy homeostasis universal to hyperphagia‐associated childhood‐onset obesity.

Key points
The cardiac energy metabolites such as ATP, phosphocreatine, ADP and NADH are kept relatively constant during physiological cardiac workload transition.
How this is accomplished is not yet clarified, though Ca2+ has been suggested to be one of the possible mechanisms.
We constructed a detailed mathematical model of cardiac mitochondria based on experimental data and studied whether known Ca2+‐dependent regulation mechanisms play roles in the metabolite constancy.
Model simulations revealed that the Ca2+‐dependent regulation mechanisms have important roles under the in vitro condition of isolated mitochondria where malate and glutamate were mitochondrial substrates, while they have only a minor role and the composition of substrates has marked influence on the metabolite constancy during workload transition under the simulated in vivo condition where many substrates exist.
These results help us understand the regulation mechanisms of cardiac energy metabolism during physiological cardiac workload transition.
Abstract
The cardiac energy metabolites such as ATP, phosphocreatine, ADP and NADH are kept relatively constant over a wide range of cardiac workload, though the mechanisms are not yet clarified. One possible regulator of mitochondrial metabolism is Ca2+, because it activates several mitochondrial enzymes and transporters. Here we constructed a mathematical model of cardiac mitochondria, including oxidative phosphorylation, substrate metabolism and ion/substrate transporters, based on experimental data, and studied whether the Ca2+‐dependent activation mechanisms play roles in metabolite constancy. Under the in vitro condition of isolated mitochondria, where malate and glutamate were used as mitochondrial substrates, the model well reproduced the Ca2+ and inorganic phosphate (Pi) dependences of oxygen consumption, NADH level and mitochondrial membrane potential. The Ca2+‐dependent activations of the aspartate/glutamate carrier and the F1Fo‐ATPase, and the Pi‐dependent activation of Complex III were key factors in reproducing the experimental data. When the mitochondrial model was implemented in a simple cardiac cell model, simulation of workload transition revealed that cytoplasmic Ca2+ concentration ([Ca2+]cyt) within the physiological range markedly increased NADH level. However, the addition of pyruvate or citrate attenuated the Ca2+ dependence of NADH during the workload transition. Under the simulated in vivo condition where malate, glutamate, pyruvate, citrate and 2‐oxoglutarate were used as mitochondrial substrates, the energy metabolites were more stable during the workload transition and NADH level was almost insensitive to [Ca2+]cyt. It was revealed that mitochondrial substrates have a significant influence on metabolite constancy during cardiac workload transition, and Ca2+ has only a minor role under physiological conditions.

Key points
Spinocerebellar ataxia type 6 (SCA6) is a midlife‐onset neurodegenerative disease caused by a CACNA1A mutation; CACNA1A is also implicated in cerebellar development.
We have previously shown that when disease symptoms are present in midlife in SCA684Q/84Q mice, cerebellar Purkinje cells spike with reduced rate and precision. In contrast, we find that during postnatal development (P10–13), SCA684Q/84Q Purkinje cells spike with elevated rate and precision.
Although surplus climbing fibres are linked to ataxia in other mouse models, we found surplus climbing fibre inputs on developing (P10–13) SCA684Q/84Q Purkinje cells when motor deficits were not detected.
Developmental alterations were transient and were no longer observed in weanling (P21–24) SCA684Q/84Q Purkinje cells.
Our results suggest that changes in the developing cerebellar circuit can occur without detectable motor abnormalities, and that changes in cerebellar development may not necessarily persist into adulthood.
Abstract
Although some neurodegenerative diseases are caused by mutations in genes that are known to regulate neuronal development, surprisingly, patients may not present disease symptoms until adulthood. Spinocerebellar ataxia type 6 (SCA6) is one such midlife‐onset disorder in which the mutated gene, CACNA1A, is implicated in cerebellar development. We wondered whether changes were observed in the developing cerebellum in SCA6 prior to the detection of motor deficits. To address this question, we used a transgenic mouse with a hyper‐expanded triplet repeat (SCA684Q/84Q) that displays late‐onset motor deficits at 7 months, and measured cerebellar Purkinje cell synaptic and intrinsic properties during postnatal development. We found that firing rate and precision were enhanced during postnatal development in P10–13 SCA684Q/84Q Purkinje cells, and observed surplus multiple climbing fibre innervation without changes in inhibitory input or dendritic structure during development. Although excess multiple climbing fibre innervation has been associated with ataxic symptoms in several adult transgenic mice, we observed no detectable changes in cerebellar‐related motor behaviour in developing SCA684Q/84Q mice. Interestingly, we found that developmental alterations were transient, as both Purkinje cell firing properties and climbing fibre innervation from weanling‐aged (P21–24) SCA684Q/84Q mice were indistinguishable from litter‐matched control mice. Our results demonstrate that significant alterations in neuronal circuit development may be observed without any detectable behavioural read‐out, and that early changes in brain development may not necessarily persist into adulthood in midlife‐onset diseases.

Key points
Hypothalamic proopiomelanocortin (POMC) neurons release peptide products that potently inhibit food intake and reduce body weight.
These neurons also release the amino acid transmitter GABA, which can inhibit downstream neurons.
Although the release of peptide transmitters from POMC neurons is regulated by energy state, whether similar regulation of GABA release might occur had not been examined.
The present results show that the GABAergic phenotype of POMC neurons is decreased selectively by caloric deficit and not altered by high‐fat diet or stress.
The fact the GABAergic phenotype of POMC neurons is sensitive to energy state suggests a dynamic physiological role for this transmitter and highlights the importance of determining the functional consequence of GABA released from POMC neurons in terms of the regulation of normal energy balance.
Abstract
In addition to peptide transmitters, hypothalamic neurons, including proopiomelanocortin (POMC) and agouti‐related peptide (AgRP) neurons, also release amino acid transmitters that can alter energy balance regulation. While recent studies show that the GABAergic nature of AgRP neurons is increased by caloric restriction, whether the GABAergic phenotype of POMC neurons is also regulated in an energy‐state‐dependent manner has not been previously examined. The present studies used fluorescence in situ hybridization to detect Gad1 and Gad2 mRNA in POMC neurons, as these encode the glutamate decarboxylase enzymes GAD67 and GAD65, respectively. The results show that both short‐term fasting and chronic caloric restriction significantly reduce the percentage of POMC neurons expressing Gad1 mRNA in both male and female mice, with less of an effect on Gad2 expression. Neither acute nor chronic intermittent restraint stress altered Gad1 expression in POMC neurons. Maintenance on a high‐fat diet also did not affect the portion POMC neurons expressing Gad1, suggesting that the GABAergic phenotype of POMC neurons is particularly sensitive to energy deficit. Because changes in Gad1 expression have been previously shown to correlate with altered terminal GABA release, fasting is likely to cause a decrease in GABA release from POMC neurons. Altogether, the present results show that the GABAergic nature of POMC neurons can be dynamically regulated by energy state in a manner opposite to that in AgRP neurons and suggest the importance of considering the functional role of GABA release in addition to the peptide transmitters from POMC neurons.

Key points
Oxytocin release from the posterior pituitary gland stimulates uterine contraction during birth but the central mechanisms that activate oxytocin neurones for birth are not well characterized.
We found that that kisspeptin fibre density around oxytocin neurones increases in late‐pregnant rats.
These kisspeptin fibres originated from hypothalamic periventricular nucleus neurones that upregulated kisspeptin expression in late pregnancy.
Oxytocin neurones were excited by central kisspeptin administration in late‐pregnant rats but not in non‐pregnant rats or early‐ to mid‐pregnant rats.
Our results reveal the emergence of a new excitatory kisspeptin projection to the oxytocin system in late pregnancy that might contribute to oxytocin neurone activation for birth.
Abstract
The hormone oxytocin promotes uterine contraction during parturition. Oxytocin is synthesized by magnocellular neurones in the hypothalamic supraoptic and paraventricular nuclei and is released into the circulation from the posterior pituitary gland in response to action potential firing. Systemic kisspeptin administration increases oxytocin neurone activity to elevate plasma oxytocin levels. Here, immunohistochemistry revealed that rats on the expected day of parturition (day 21 of gestation) had a higher density of kisspeptin‐positive fibres in the perinuclear zone surrounding the supraoptic nucleus (which provides dense glutamatergic and GABAergic innervation to the supraoptic nucleus) than was evident in non‐pregnant rats. Retrograde tracing showed the kisspeptin projections to the perinuclear zone originated from the hypothalamic periventricular nucleus. Quantitative RT‐PCR showed that kisspeptin receptor mRNA, Kiss1R mRNA, was expressed in the perinuclear zone–supraoptic nucleus and that the relative Kiss1R mRNA expression does not change over the course of pregnancy. Finally, intracerebroventricular administration of kisspeptin increased the firing rate of oxytocin neurones in anaesthetized late‐pregnant rats (days 18–21 of gestation) but not in non‐pregnant rats, or in early‐ or mid‐pregnant rats. Taken together, these results suggest that kisspeptin expression is upregulated in the periventricular nucleus projection to the perinuclear zone of the supraoptic nucleus towards the end of pregnancy. Hence, this input might activate oxytocin neurones during parturition.

Key points
The effects of short‐term (ST; 10 days) and long‐term (LT; 30 days) intermittent hypoxia (IH) on blood pressure (BP), breathing and carotid body (CB) chemosensory reflex were examined in adult rats.
ST‐ and LT‐IH treated rats exhibited hypertension, irregular breathing with apnoea and augmented the CB chemosensory reflex, with all these responses becoming normalized during recovery from ST‐ but not from LT‐IH.
The persistent cardiorespiratory responses to LT‐IH were associated with elevated reactive oxygen species (ROS) levels in the CB and adrenal medulla, which were a result of DNA methylation‐dependent suppression of genes encoding anti‐oxidant enzymes (AOEs).
Treating rats with decitabine either during LT‐IH or during recovery from LT‐IH prevented DNA methylation of AOE genes, normalized the expression of AOE genes and ROS levels, reversed the heightened CB chemosensory reflex and hypertension, and also stabilized breathing.
Abstract
Rodents exposed to chronic intermittent hypoxia (IH), simulating blood O2 saturation profiles during obstructive sleep apnoea (OSA), have been shown to exhibit a heightened carotid body (CB) chemosensory reflex and hypertension. CB chemosensory reflex activation also results in unstable breathing with apnoeas. However, the effect of chronic IH on breathing is not known. In the present study, we examined the effects of chronic IH on breathing along with blood pressure (BP) and assessed whether the autonomic responses are normalized after recovery from chronic IH. Studies were performed on adult, male, Sprague–Dawley rats exposed to either short‐term (ST; 10 days) or long‐term (LT, 30 days) IH. Rats exposed to either ST‐ or LT‐IH exhibited hypertension, irregular breathing with apnoeas, an augmented CB chemosensory reflex as indicated by elevated CB neural activity and plasma catecholamine levels, and elevated reactive oxygen species (ROS) levels in the CB and adrenal medulla (AM). All these effects were normalized after recovery from ST‐IH but not from LT‐IH. Analysis of the molecular mechanisms underlying the persistent effects of LT‐IH revealed increased DNA methylation of genes encoding anti‐oxidant enzymes (AOEs). Treatment with decitabine, a DNA methylation inhibitor, either during LT‐IH or during recovery from LT‐IH, prevented DNA methylation, normalized the expression of AOE genes, ROS levels, CB chemosensory reflex and BP, and also stabilized breathing. These results suggest that persistent cardiorespiratory abnormalities caused by LT‐IH are mediated by epigenetic re‐programming of the redox state in the CB chemosensory reflex pathway.

Key points
Neuroinflammation associated with CNS insults leads to neuronal hyperexcitability, which may culminate in epileptiform discharges.
Application of the endotoxin lipopolysaccharide (LPS) to brain tissue initiates a neuroinflammatory cascade, providing an experimental model to study the mechanisms of neuroinflammatory neuronal hyperexcitability.
Here we show that LPS application to hippocampal slices markedly enhances the excitability of CA1 pyramidal cells by inhibiting a specific potassium current, the M‐current, generated by KV7/M channels, which controls the excitability of almost every neuron in the CNS.
The LPS‐induced M‐current inhibition is triggered by sequential activation of microglia, astrocytes and pyramidal cells, mediated by metabotropic purinergic and glutamatergic transmission, leading to blockade of KV7/M channels by calcium released from intracellular stores.
The identification of the downstream molecular target of neuroinflammation, namely the KV7/M channel, potentially has far reaching implications for the understanding and treatment of many acute and chronic brain disorders.
Abstract
Acute brain insults and many chronic brain diseases manifest an innate inflammatory response. The hallmark of this response is glia activation, which promotes repair of damaged tissue, but also induces structural and functional changes that may lead to an increase in neuronal excitability. We have investigated the mechanisms involved in the modulation of neuronal activity by acute inflammation. Initiating inflammatory responses in hippocampal tissue rapidly led to neuronal depolarization and repetitive firing even in the absence of active synaptic transmission. This action was mediated by a complex metabotropic purinergic and glutamatergic glia‐to‐neuron signalling cascade, leading to the blockade of neuronal KV7/M channels by Ca2+ released from internal stores. These channels generate the low voltage‐activating, non‐inactivating M‐type K+ current (M‐current) that controls intrinsic neuronal excitability, and its inhibition was the predominant cause of the inflammation‐induced hyperexcitability. Our discovery that the ubiquitous KV7/M channels are the downstream target of the inflammation‐induced cascade, has far reaching implications for the understanding and treatment of many acute and chronic brain disorders.

Abstract
Computational models of many aspects of the mammalian cardiovascular circulation have been developed. Indeed, along with orthopaedics, this area of physiology is one that has attracted much interest from engineers, presumably because the equations governing blood flow in the vascular system are well understood and can be solved with well‐established numerical techniques. Unfortunately, there have been only a few attempts to create a comprehensive public domain resource for cardiovascular researchers. In this paper we propose a roadmap for developing an open source cardiovascular circulation model. The model should be registered to the musculo‐skeletal system. The computational infrastructure for the cardiovascular model should provide for near real‐time computation of blood flow and pressure in all parts of the body. The model should deal with vascular beds in all tissues, and the computational infrastructure for the model should provide links into CellML models of cell function and tissue function. In this work we review the literature associated with 1D blood flow modelling in the cardiovascular system, discuss model encoding standards, software and a model repository. We then describe the coordinate systems used to define the vascular geometry, derive the equations and discuss the implementation of these coupled equations in the open source computational software OpenCMISS. Finally, some preliminary results are presented and plans outlined for the next steps in the development of the model, the computational software and the graphical user interface for accessing the model.

Key points
The present study tested whether HCN channels contribute to the organization of motor cortex and to skilled motor behaviour during a forelimb reaching task.
Experimental reductions in HCN channel signalling increase the representation of complex multiple forelimb movements in motor cortex as assessed by intracortical microstimulation.
Global HCN1KO mice exhibit reduced reaching accuracy and atypical movements during a single‐pellet reaching task relative to wild‐type controls.
Acute pharmacological inhibition of HCN channels in forelimb motor cortex decreases reaching accuracy and increases atypical movements during forelimb reaching.
Abstract
The mechanisms by which distinct movements of a forelimb are generated from the same area of motor cortex have remained elusive. Here we examined a role for HCN channels, given their ability to alter synaptic integration, in the expression of forelimb movement responses during intracortical microstimulation (ICMS) and movements of the forelimb on a skilled reaching task. We used short‐duration high‐resolution ICMS to evoke forelimb movements following pharmacological (ZD7288), experimental (electrically induced cortical seizures) or genetic approaches that we confirmed with whole‐cell patch clamp to substantially reduce Ih current. We observed significant increases in the number of multiple movement responses evoked at single sites in motor maps to all three experimental manipulations in rats or mice. Global HCN1 knockout mice were less successful and exhibited atypical movements on a skilled‐motor learning task relative to wild‐type controls. Furthermore, in reaching‐proficient rats, reaching accuracy was reduced and forelimb movements were altered during infusion of ZD7288 within motor cortex. Thus, HCN channels play a critical role in the separation of overlapping movement responses and allow for successful reaching behaviours. These data provide a novel mechanism for the encoding of multiple movement responses within shared networks of motor cortex. This mechanism supports a viewpoint of primary motor cortex as a site of dynamic integration for behavioural output.

Key points
High‐intensity interval training (HIIT) has become popular, time‐sparing alternative to moderate intensity continuous training (MICT), although the cardiac vascular and metabolic effects of HIIT are incompletely known.
We compared the effects of 2‐week interventions with HIIT and MICT on myocardial perfusion and free fatty acid and glucose uptake.
Insulin‐stimulated myocardial glucose uptake was decreased by training without any significantly different response between the groups, whereas free fatty acid uptake remained unchanged.
Adenosine‐stimulated myocardial perfusion responded differently to the training modes (change in mean HIIT: –19%; MICT: +9%; P = 0.03 for interaction) and was correlated with myocardial glucose uptake for the entire dataset and especially after HIIT training.
HIIT and MICT induce similar metabolic and functional changes in the heart, although myocardial vascular hyperaemic reactivity is impaired after HIIT, and this should be considered when prescribing very intense HIIT for previously untrained subjects.
Abstract
High‐intensity interval training (HIIT) is a time‐efficient way of obtaining the health benefits of exercise, although the cardiac effects of this training mode are incompletely known. We compared the effects of short‐term HIIT and moderate intensity continuous training (MICT) interventions on myocardial perfusion and metabolism and cardiac function in healthy, sedentary, middle‐aged men. Twenty‐eight healthy, middle‐aged men were randomized to either HIIT or MICT groups (n = 14 in both) and underwent six cycle ergometer training sessions within 2 weeks (HIIT session: 4–6 × 30 s all‐out cycling/4 min recovery, MICT session 40–60 min at 60% V̇O2 peak ). Cardiac magnetic resonance imaging (CMRI) was performed to measure cardiac structure and function and positron emission tomography was used to measure myocardial perfusion at baseline and during adenosine stimulation, insulin‐stimulated glucose uptake (MGU) and fasting free fatty acid uptake (MFFAU). End‐diastolic and end‐systolic volumes increased and ejection fraction slightly decreased with both training modes, although no other changes in CMRI were observed. MFFAU and basal myocardial perfusion remained unchanged. MGU was decreased by training (HIIT from 46.5 to 35.9; MICT from 47.4 to 44.4 mmol 100 g–1 min–1, P = 0.007 for time, P = 0.11 for group × time). Adenosine‐stimulated myocardial perfusion responded differently to the training modes (change in mean HIIT: –19%; MICT: +9%; P = 0.03 for group × time interaction). HIIT and MICT induce similar metabolic and functional changes in the heart, although myocardial vascular hyperaemic reactivity is impaired after HIIT. This should be taken into account when prescribing very intense HIIT for previously untrained subjects.

Key points
Cognitive performance is impaired by hypoxia despite global cerebral oxygen delivery and metabolism being maintained.
Using arterial spin labelled (ASL) magnetic resonance imaging, this is the first study to show regional reductions in cerebral blood flow (CBF) in response to decreased oxygen supply (hypoxia) at 2 h that increased in area and became more pronounced at 10 h.
Reductions in CBF were seen in brain regions typically associated with the ‘default mode’ or ‘task negative’ network.
Regional reductions in CBF, and associated vasoconstriction, within the default mode network in hypoxia is supported by increased vasodilatation in these regions to a subsequent hypercapnic (5% CO2) challenge.
These results suggest an anatomical mechanism through which hypoxia may cause previously reported deficits in cognitive performance.
Abstract
Hypoxia causes an increase in global cerebral blood flow, which maintains global cerebral oxygen delivery and metabolism. However, neurological deficits are abundant under hypoxic conditions. We investigated regional cerebral microvascular responses to acute (2 h) and prolonged (10 h) poikilocapnic normobaric hypoxia. We found that 2 h of hypoxia caused an expected increase in frontal cortical grey matter perfusion but unexpected perfusion decreases in regions of the brain normally associated with the ‘default mode’ or ‘task negative’ network. After 10 h in hypoxia, decreased blood flow to the major nodes of the default mode network became more pronounced and widespread. The use of a hypercapnic challenge (5% CO2) confirmed that these reductions in cerebral blood flow from hypoxia were related to vasoconstriction. Our findings demonstrate steady‐state deactivation of the default network under acute hypoxia, which become more pronounced over time. Moreover, these data provide a unique insight into the nuanced localized cerebrovascular response to hypoxia that is not attainable through traditional methods. The observation of reduced perfusion in the posterior cingulate and cuneal cortex, which are regions assumed to play a role in declarative and procedural memory, provides an anatomical mechanism through which hypoxia may cause deficits in working memory.

Key points
Rapid onset vasodilatation (ROV) initiates functional hyperaemia upon skeletal muscle contraction and is attenuated during ageing via α‐adrenoreceptor (αAR) stimulation, but it is unknown where this effect predominates in resistance networks.
In gluteus maximus muscles of young (4 months) and old (24 months) male C57BL/6 mice, tetanic contraction while observing feed arteries and arterioles initiated ROV, which increased with contraction duration, peaked later in upstream versus downstream vessel branches and was attenuated throughout networks with advanced age.
With no effect on muscle force production, inhibiting αARs improved ROV in old mice while activating αARs attenuated ROV in young mice.
Modulating ROV through αARs was greater in upstream feed arteries and arterioles compared to downstream arterioles, with α2ARs more effective than α1ARs.
ROV is coordinated along resistance networks and modulated differentially between young and old mice via αARs; with advanced age, attenuated dilatation of upstream branches will restrict muscle blood flow.
Abstract
Rapid onset vasodilatation (ROV) in skeletal muscle is attenuated during advanced age via α‐adrenoreceptor (αAR) activation, but it is unknown where such effects predominate in the resistance vasculature. Studying the gluteus maximus muscle (GM) of anaesthetized young (4 months) and old (24 months) male C57BL/6 mice, we tested the hypothesis that attenuation of ROV during advanced age is most effective in proximal branches of microvascular resistance networks. Diameters of a feed artery (FA) and first‐ (1A), second‐ (2A) and third‐ (3A) order arterioles were studied in response to single tetanic contractions (100 Hz, 100–1000 ms). ROV began within 1 s and peaked sooner in 2A and 3A (∼3 s) than in 1A or FA (∼4 s). Relative amplitudes of dilatation increased with contraction duration and with vessel branch order (FA<1A<2A<3A). In old mice, attenuation of ROV was greater in FA and 1A compared to 2A and 3A. With no effect on muscle force production, inhibiting αARs (phentolamine; 10−6 m) improved ROV in FA and 1A of old mice while subthreshold stimulation of αARs in young mice (noradrenaline; 10−9 m) depressed ROV most effectively in FA and 1A. In young mice, stimulating α1ARs (phenylephrine; 10−7 m) and α2ARs (UK 14304; 10−7 m) attenuated ROV primarily in FA. In old mice, inhibiting α2ARs (rauwolscine; 10−7 m) restored ROV more effectively in FA and 1A than did inhibiting α1ARs (prazosin; 10−8 m). We conclude that, with temporal and spatial coordination along resistance networks, attenuation of ROV with advanced age is most effective in proximal branches via constitutive activation of α2ARs.

Key points
In young adults, cyclooxygenase (COX) contributes to the heat loss responses of cutaneous vasodilatation and sweating, and this may be mediated by prostacyclin‐induced activation of nitric oxide synthase (NOS) and calcium‐activated potassium (KCa) channels.
This prostacyclin‐induced response may be diminished in older relative to young adults because ageing is known to attenuate COX‐dependent heat loss responses.
We observed that, although prostacyclin does not mediate sweating in young and older males, it does modulate cutaneous vasodilatation, although the magnitude of increase is similar between groups.
We also found that, although NOS and KCa channels contribute to prostacyclin‐induced cutaneous vasodilatation in young males, these contributions are diminished in older males.
Our findings provide new insight into the mechanisms governing heat loss responses and suggest that the age‐related diminished COX‐dependent heat loss responses reported in previous studies may be a result of the reduced COX‐derived production of prostanoids (e.g., prostacyclin) rather than the decreased sensitivity of prostanoid receptors.
Abstract
Cyclooxygenase (COX) contributes to the regulation of cutaneous vasodilatation and sweating; however, the mechanism(s) underpinning this response remain unresolved. We hypothesized that prostacyclin (a COX‐derived product) may directly mediate cutaneous vasodilatation and sweating through nitric oxide synthase (NOS) and calcium‐activated potassium (KCa) channels in young adults. However, these responses would be diminished in older adults because ageing attenuates COX‐dependent cutaneous vasodilatation and sweating. In young (25 ± 4 years) and older (60 ± 6 years) males (nine per group), cutaneous vascular conductance (CVC) and sweat rate were evaluated at four intradermal forearm skin sites: (i) control; (ii) 10 mm NG‐nitro‐l‐arginine (l‐NNA), a non‐specific NOS inhibitor; (iii) 50 mm tetraethylammonium (TEA), a non‐specific KCa channel blocker; and (iv) 10 mm l‐NNA + 50 mm TEA. All four sites were coadministered with prostacyclin in an incremental manner (0.04, 0.4, 4, 40 and 400 μm each for 25 min). Prostacyclin‐induced increases in CVC were similar between groups (all concentrations, P > 0.05). l‐NNA and TEA, as well as their combination, lowered CVC in young males at all prostacyclin concentrations (P ≤ 0.05), with the exception of l‐NNA at 0.04 μm (P > 0.05). In older males, CVC during prostacyclin administration was not influenced by l‐NNA (all concentrations), TEA (4‐400 μm) or their combination (400 μm) (P > 0.05). No effect on sweat rate was observed in either group (all concentrations, P > 0.05). We conclude that, although prostacyclin does not mediate sweating, it modulates cutaneous vasodilatation to a similar extent in young and older males. Furthermore, although NOS and KCa channels contribute to the prostacyclin‐induced cutaneous vasodilatation in young males, these contributions are diminished in older males.

Key points
Retigabine is a KCNQ voltage‐gated potassium channel opener that was recently approved as an add‐on therapeutic for patients with drug‐resistant epilepsy.
Retigabine exhibits very little specificity between most KCNQ channel subtypes, and there is interest in generating more potent and specific KCNQ channel openers.
The present study describes the marked specificity of ICA069673 for KCNQ2 vs. KCNQ3, and exploits this property to investigate determinants of KCNQ subtype specificity.
ICA069673 acts on a binding site in the voltage‐sensing domain that is distinct from the putative retigabine site in the channel pore.
ICA069673 has two separable effects on KCNQ channel activity. We identify two channel residues required for subtype specificity of KCNQ channel openers and show that these are sufficient to generate ICA069673 sensitivity in KCNQ3.
Abstract
Retigabine (RTG) is the first approved anti‐epileptic drug that acts via activation of voltage‐gated potassium channels, targeting KCNQ channels that underlie the neuronal M‐current. RTG exhibits little specificity between KCNQ2–5 as a result of conservation of a Trp residue in the pore domain that binds to the drug. The RTG analogue ICA‐069673 (‘ICA73’) exhibits much stronger effects on KCNQ2 channels, including a large hyperpolarizing shift of the voltage‐dependence of activation, an ∼2‐fold enhancement of peak current and pronounced subtype specificity for KCNQ2 over KCNQ3. Based on ICA73 sensitivity of chimeric constructs of the transmembrane segments of KCNQ2 and KCNQ3, this drug appears to interact with the KCNQ2 voltage sensor (S1–S4) rather than the pore region targeted by RTG. KCNQ2 point mutants in the voltage sensor were generated based on KCNQ2/KCNQ3 sequence differences, and screened for ICA73 sensitivity. These experiments reveal that KCNQ2 residues F168 and A181 in the S3 segment are essential determinants of ICA73 subtype specificity. Mutations at either position in KCNQ2 abolish the ICA73‐mediated gating shift, but preserve RTG sensitivity. Interestingly, A181P mutant channels show little ICA73‐mediated gating shift but retain current potentiation by the drug. Mutations (L198F and P211A), which introduce these critical KCNQ2 residues at corresponding positions in KCNQ3, transplant partial ICA73 sensitivity. These findings demonstrate that RTG and ICA73 act via distinct mechanisms, and also reveal specific residues that underlie subtype specificity of KCNQ channel openers.

Key points
Agonist‐dependent oscillations in the concentration of free cytosolic calcium are a vital mechanism for the control of airway smooth muscle contraction and thus are a critical factor in airway hyper‐responsiveness.
Using a mathematical model, closely tied to experimental work, we show that the oscillations in membrane potential accompanying the calcium oscillations have no significant effect on the properties of the calcium oscillations.
In addition, the model shows that calcium entry through store‐operated calcium channels is critical for calcium oscillations, but calcium entry through voltage‐gated channels has much less effect.
The model predicts that voltage‐gated channels are less important than store‐operated channels in the control of airway smooth muscle tone.
Abstract
Airway smooth muscle contraction is typically the key mechanism underlying airway hyper‐responsiveness, and the strength of muscle contraction is determined by the frequency of oscillations of intracellular calcium (Ca2+) concentration. In airway smooth muscle cells, these Ca2+ oscillations are caused by cyclic Ca2+ release from the sarcoplasmic reticulum, although Ca2+ influx via plasma membrane channels is also necessary to sustain the oscillations over longer times. To assess the relative contributions of store‐operated and voltage‐gated Ca2+ channels to this Ca2+ influx, we generated a comprehensive mathematical model, based on experimental Ca2+ measurements in mouse precision‐cut lung slices, to simulate Ca2+ oscillations and changes in membrane potential. Agonist‐induced Ca2+ oscillations are accompanied by oscillations in membrane potential, although the membrane potential oscillations are too small to generate large Ca2+ currents through voltage‐gated Ca2+ channels, and thus have little effect on the Ca2+ oscillations. Ca2+ entry through voltage‐gated channels only becomes important when the cell is depolarized (e.g. by a high external K+ concentration). As a result, agonist‐induced Ca2+ oscillations are critically dependent on Ca2+ entry through store‐operated channels but do not depend strongly on Ca2+ entry though voltage‐gated channels.

Key points
Motor neurons are the output neurons of the central nervous system and are responsible for controlling muscle contraction.
When initially activated during voluntary contraction, firing rates of motor neurons increase steeply but then level out at modest rates.
Activation of an intrinsic source of excitatory current at recruitment onset may underlie the initial steep increase in firing rate in motor neurons.
We attempted to disable this intrinsic excitatory current by artificially activating an inhibitory reflex.
When motor neuron activity was recorded while the inhibitory reflex was engaged, firing rates no longer increased steeply, suggesting that the intrinsic excitatory current was probably responsible for the initial sharp rise in motor neuron firing rate.
Abstract
During graded isometric contractions, motor unit (MU) firing rates increase steeply upon recruitment but then level off at modest rates even though muscle force continues to increase. The mechanisms underlying such firing behaviour are not known although activation of persistent inward currents (PICs) might be involved. PICs are intrinsic, voltage‐dependent currents that activate strongly when motor neurons (MNs) are first recruited. Such activation might cause a sharp escalation in depolarizing current and underlie the steep initial rise in MU firing rate. Because PICs can be disabled with synaptic inhibition, we hypothesized that artificial activation of an inhibitory pathway might curb this initial steep rise in firing rate. To test this, human subjects performed slow triangular ramp contractions of the ankle dorsiflexors in the absence and presence of tonic synaptic inhibition delivered to tibialis anterior (TA) MNs by sural nerve stimulation. Firing rate profiles (expressed as a function of contraction force) of TA MUs recorded during these tasks were compared for control and stimulation conditions. Under control conditions, during the ascending phase of the triangular contractions, 93% of the firing rate profiles were best fitted by rising exponential functions. With stimulation, however, firing rate profiles were best fitted with linear functions or with less steeply rising exponentials. Firing rate profiles for the descending phases of the contractions were best fitted with linear functions for both control and stimulation conditions. These results seem consistent with the idea that PICs contribute to non‐linear firing rate profiles during ascending but not descending phases of contractions.

Key points
The short‐chain fatty acids (SCFAs) are bacterial metabolites produced during the colonic fermentation of undigested carbohydrates, such as dietary fibre and prebiotics, and can mediate the interaction between the diet, the microbiota and the host.
We quantified the fraction of colonic administered SCFAs that could be recovered in the systemic circulation, the fraction that was excreted via the breath and urine, and the fraction that was used as a precursor for glucose, cholesterol and fatty acids.
This information is essential for understanding the molecular mechanisms by which SCFAs beneficially affect physiological functions such as glucose and lipid metabolism and immune function.
Abstract
The short‐chain fatty acids (SCFAs), acetate, propionate and butyrate, are bacterial metabolites that mediate the interaction between the diet, the microbiota and the host. In the present study, the systemic availability of SCFAs and their incorporation into biologically relevant molecules was quantified. Known amounts of 13C‐labelled acetate, propionate and butyrate were introduced in the colon of 12 healthy subjects using colon delivery capsules and plasma levels of 13C‐SCFAs 13C‐glucose, 13C‐cholesterol and 13C‐fatty acids were measured. The butyrate‐producing capacity of the intestinal microbiota was also quantified. Systemic availability of colonic‐administered acetate, propionate and butyrate was 36%, 9% and 2%, respectively. Conversion of acetate into butyrate (24%) was the most prevalent interconversion by the colonic microbiota and was not related to the butyrate‐producing capacity in the faecal samples. Less than 1% of administered acetate was incorporated into cholesterol and <15% in fatty acids. On average, 6% of colonic propionate was incorporated into glucose. The SCFAs were mainly excreted via the lungs after oxidation to 13CO2, whereas less than 0.05% of the SCFAs were excreted into urine. These results will allow future evaluation and quantification of SCFA production from 13C‐labelled fibres in the human colon by measurement of 13C‐labelled SCFA concentrations in blood.

Key points
The degree to which developmental programmes or environmental signals determine physiological phenotypes remains a major question in physiology.
Vertebrates change environments during development, confounding interpretation of the degree to which development (i.e. permanent processes) or phenotypic plasticity (i.e. reversible processes) produces phenotypes.
Tadpoles mainly breathe water for gas exchange and frogs may breathe water or air depending on their environment and are, therefore, exemplary models to differentiate the degree to which life‐stage vs. environmental context drives developmental phenotypes associated with neural control of lung breathing.
Using isolated brainstem preparations and patch clamp electrophysiology, we demonstrate that adult bullfrogs acclimatized to water‐breathing conditions do not exhibit CO2 and O2 chemosensitivity of lung breathing, similar to water‐breathing tadpoles.
Our results establish that phenotypes associated with developmental stage may arise from plasticity per se and suggest that a developmental trajectory coinciding with environmental change obscures origins of stage‐dependent physiological phenotypes by masking plasticity.
Abstract
An unanswered question in developmental physiology is to what extent does the environment vs. a genetic programme produce phenotypes? Developing animals inhabit different environments and switch from one to another. Thus a developmental time course overlapping with environmental change confounds interpretations as to whether development (i.e. permanent processes) or phenotypic plasticity (i.e. reversible processes) generates phenotypes. Tadpoles of the American bullfrog, Lithobates catesbeianus, breathe water at early life‐stages and minimally use lungs for gas exchange. As adults, bullfrogs rely on lungs for gas exchange, but spend months per year in ice‐covered ponds without lung breathing. Aquatic submergence, therefore, removes environmental pressures requiring lung breathing and enables separation of adulthood from environmental factors associated with adulthood that necessitate control of lung ventilation. To test the hypothesis that postmetamorphic respiratory control phenotypes arise through permanent developmental changes vs. reversible environmental signals, we measured respiratory‐related nerve discharge in isolated brainstem preparations and action potential firing from CO2‐sensitive neurons in bullfrogs acclimatized to semi‐terrestrial (air‐breathing) and aquatic‐overwintering (no air‐breathing) habitats. We found that aquatic overwintering significantly reduced neuroventilatory responses to CO2 and O2 involved in lung breathing. Strikingly, this gas sensitivity profile reflects that of water‐breathing tadpoles. We further demonstrated that aquatic overwintering reduced CO2‐induced firing responses of chemosensitive neurons. In contrast, respiratory rhythm generating processes remained adult‐like after submergence. Our results establish that phenotypes associated with life‐stage can arise from phenotypic plasticity per se. This provides evidence that developmental time courses coinciding with environmental changes obscure interpretations regarding origins of stage‐dependent physiological phenotypes by masking plasticity.

Key points
Using in vivo electrophysiology, we find that a subset of whisker‐responsive neurons in the ventral posterior medial region (VPM) respond to visual stimuli.
These light‐responsive neurons in the VPM are particularly sensitive to optic flow.
Presentation of optic flow stimuli modulates the amplitude of concurrent whisker responses.
Visual information reaches the VPM via a circuit encompassing the visual cortex.
These data represent a new example of cross‐modal integration in the primary sensory thalamus.
Abstract
Sensory signals reach the cortex via sense‐specific thalamic nuclei. Here we report that neurons in the primary sensory thalamus of the mouse vibrissal system (the ventral posterior medial region; VPM) can be excited by visual as well as whisker stimuli. Using extracellular electrophysiological recordings from anaesthetized mice we first show that simple light steps can excite a subset of VPM neurons. We then test the ability of the VPM to respond to spatial patterns and show that many units are excited by visual motion in a direction‐selective manner. Coherent movement of multiple objects (an artificial recreation of ‘optic flow’ that would usually occur during head rotations or body movements) best engages this visual motion response. We next show that, when co‐applied with visual stimuli, the magnitude of responses to whisker deflections is highest in the presence of optic flow going in the opposite direction. Importantly, whisker response amplitude is also modulated by presentation of a movie recreating the mouse's visual experience during natural exploratory behaviour. We finally present functional and anatomical data indicating a functional connection (probably multisynaptic) from the primary visual cortex to VPM. These data provide a rare example of multisensory integration occurring at the level of the sensory thalamus, and provide evidence for dynamic regulation of whisker responses according to visual experience.

Key points
The midbrain periaqueductal grey (PAG) forms part of an endogenous analgesic system which is tightly regulated by the neurotransmitter GABA.
The role of endocannabinoids in regulating GABAergic control of this system was examined in rat PAG slices.
Under basal conditions GABAergic neurotransmission onto PAG output neurons was multivesicular.
Activation of the endocannabinoid system reduced GABAergic inhibition by reducing the probability of release and by shifting release to a univesicular mode.
Blockade of endocannabinoid system unmasked a tonic control over the probability and mode of GABA release.
These findings provides a mechanistic foundation for the control of the PAG analgesic system by disinhibition.
Abstract
The midbrain periaqueductal grey (PAG) has a crucial role in coordinating endogenous analgesic responses to physiological and psychological stressors. Endocannabinoids are thought to mediate a form of stress‐induced analgesia within the PAG by relieving GABAergic inhibition of output neurons, a process known as disinhibition. This disinhibition is thought to be achieved by a presynaptic reduction in GABA release probability. We examined whether other mechanisms have a role in endocannabinoid modulation of GABAergic synaptic transmission within the rat PAG. The group I mGluR agonist DHPG ((R,S)‐3,5‐dihydroxyphenylglycine) inhibited evoked IPSCs and increased their paired pulse ratio in normal external Ca2+, and when release probability was reduced by lowering Ca2+. However, the effect of DHPG on the coefficient of variation and kinetics of evoked IPSCs differed between normal and low Ca2+. Lowering external Ca2+ had a similar effect on evoked IPSCs to that observed for DHPG in normal external Ca2+. The low affinity GABAA receptor antagonist TPMPA ((1,2,5,6‐tetrahydropyridin‐4‐yl)methylphosphinic acid) inhibited evoked IPSCs to a greater extent in low than in normal Ca2+. Together these findings indicate that the normal mode of GABA release is multivesicular within the PAG, and that DHPG and lowering external Ca2+ switch this to a univesicular mode. The effects of DHPG were mediated by mGlu5 receptor engagement of the retrograde endocannabinoid system. Blockade of endocannabinoid breakdown produced a similar shift in the mode of release. We conclude that endocannabinoids control both the mode and the probability of GABA release within the PAG.

Key points
Physical inactivity, which drastically increases with advancing age, is associated with numerous chronic diseases.
The nucleus accumbens (the pleasure and reward ‘hub’ in the brain) influences wheel running behaviour in rodents.
RNA‐sequencing and subsequent bioinformatics analysis led us to hypothesize a potential relationship between the regulation of dendritic spine density, the molecules involved in synaptic transmission, and age‐related reductions in wheel running. Upon completion of follow‐up studies, we developed the working model that synaptic plasticity in the nucleus accumbens is central to age‐related changes in voluntary running.
Testing this hypothesis, inhibition of Cdk5 (comprising a molecule central to the processes described above) in the nucleus accumbens reduced wheel running.
The results of the present study show that reductions in synaptic transmission and Cdk5 function are related to decreases in voluntary running behaviour and provide guidance for understanding the neural mechanisms that underlie age‐dependent reductions in the motivation to be physically active.
Abstract
Increases in age are often associated with reduced levels of physical activity, which, in turn, associates with the development of numerous chronic diseases. We aimed to assess molecular differences in the nucleus accumbens (NAc) (a specific brain nucleus postulated to influence rewarding behaviour) with respect to wheel running and sedentary female Wistar rats at 8 and 14 weeks of age. RNA‐sequencing was used to interrogate transcriptomic changes between 8‐ and 14‐week‐old wheel running rats, and select transcripts were later analysed by quantitative RT‐PCR in age‐matched sedentary rats. Voluntary wheel running was greatest at 8 weeks and had significantly decreased by 12 weeks. From 619 differentially expressed mRNAs, bioinformatics suggested that cAMP‐mediated signalling, dopamine‐ and cAMP‐regulated neuronal phosphoprotein of 32 kDa feedback, and synaptic plasticity were greater in 8‐ vs. 14‐week‐old rats. In depth analysis of these networks showed significant (∼20–30%; P

Key points
Neurons in the medial nucleus of the trapezoid body of anaesthetized rats of postnatal day (P)2–6 showed burst firing with a preferred interval of about 100 ms, which was stable, and a second preferred interval of 5–30 ms, which shortened during development.
In 3 out of 132 cases, evidence for the presence of two large inputs was found.
In vivo whole‐cell recordings revealed that the excitability of the principal neuron and the size of its largest synaptic inputs were developmentally matched.
At P2–4, action potentials were triggered by barrages of small synaptic events that summated to plateau potentials, while at later stages firing depended on a single, large and often prespike‐associated input, which is probably the nascent calyx of Held.
Simulations with a Hodgkin–Huxley‐like model, which was based on fits of the intrinsic postsynaptic properties, suggested an essential role for the low‐threshold potassium conductance in this transition.
Abstract
In the adult, principal neurons of the medial nucleus of the trapezoid body (MNTB) are typically contacted by a single, giant terminal called the calyx of Held, whereas during early development a principal neuron receives inputs from many axons. How these changes in innervation impact the postsynaptic activity has not yet been studied in vivo. We therefore recorded spontaneous inputs and intrinsic properties of principal neurons in anaesthetized rat pups during the developmental period in which the calyx forms. A characteristic bursting pattern could already be observed at postnatal day (P)2, before formation of the calyx. At this age, action potentials (APs) were triggered by barrages of summating EPSPs causing plateau depolarizations. In contrast, at P5, a single EPSP reliably triggered APs, resulting in a close match between pre‐ and postsynaptic firing. Postsynaptic excitability and the size of the largest synaptic events were developmentally matched. The developmental changes in intrinsic properties were estimated by fitting in vivo current injections to a Hodgkin–Huxley‐type model of the principal neuron. Our simulations indicated that the developmental increases in Ih, low‐threshold K+ channels and leak currents contributed to the reduction in postsynaptic excitability, but that low‐threshold K+ channels specifically functioned as a dampening influence in the near‐threshold range, thus precluding small inputs from triggering APs. Together, these coincident changes help to propagate bursting activity along the auditory brainstem, and are essential steps towards establishing the relay function of the calyx of Held synapse.

Key points
Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disease caused by a gene defect, leading to movement disorder such as cerebellar ataxia.
It remains largely unknown which functional defect contributes to the cerebellar ataxic phenotype in SCA1.
In this study, we report progressive dysfunction of metabotropic glutamate receptor (mGluR) signalling, which leads to smaller slow synaptic responses, reduced dendritic Ca2+ signals and impaired synaptic plasticity at cerebellar synapses, in the early disease stage of SCA1 model mice.
We also show that enhancement of mGluR signalling by a clinically available drug, baclofen, leads to improvement of motor performance in SCA1 mice.
SCA1 is an incurable disease with no effective treatment, and our results may provide mechanistic grounds for targeting mGluRs and a novel drug therapy with baclofen to treat SCA1 patients in the future.
Abstract
Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disease that presents with cerebellar ataxia and motor learning defects. Previous studies have indicated that the pathology of SCA1, as well as other ataxic diseases, is related to signalling pathways mediated by the metabotropic glutamate receptor type 1 (mGluR1), which is indispensable for proper motor coordination and learning. However, the functional contribution of mGluR signalling to SCA1 pathology is unclear. In the present study, we show that SCA1 model mice develop a functional impairment of mGluR signalling which mediates slow synaptic responses, dendritic Ca2+ signals, and short‐ and long‐term synaptic plasticity at parallel fibre (PF)–Purkinje cell (PC) synapses in a progressive manner from the early disease stage (5 postnatal weeks) prior to PC death. Notably, impairment of mGluR‐mediated dendritic Ca2+ signals linearly correlated with a reduction of PC capacitance (cell surface area) in disease progression. Enhancement of mGluR signalling by baclofen, a clinically available GABAB receptor agonist, led to an improvement of motor performance in SCA1 mice and the improvement lasted ∼1 week after a single application of baclofen. Moreover, the restoration of motor performance in baclofen‐treated SCA1 mice matched the functional recovery of mGluR‐mediated slow synaptic currents and mGluR‐dependent short‐ and long‐term synaptic plasticity. These results suggest that impairment of synaptic mGluR cascades is one of the important contributing factors to cerebellar ataxia in early and middle stages of SCA1 pathology, and that modulation of mGluR signalling by baclofen or other clinical interventions may be therapeutic targets to treat SCA1.

Key points
High dietary potassium (K+) intake dephosphorylates and inactivates the NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT).
Using several ex vivo models, we show that physiological changes in extracellular K+, similar to those occurring after a K+ rich diet, are sufficient to promote a very rapid dephosphorylation of NCC in native DCT cells.
Although the increase of NCC phosphorylation upon decreased extracellular K+ appears to depend on cellular Cl− fluxes, the rapid NCC dephosphorylation in response to increased extracellular K+ is not Cl−‐dependent.
The Cl−‐dependent pathway involves the SPAK/OSR1 kinases, whereas the Cl− independent pathway may include additional signalling cascades.
Abstract
A high dietary potassium (K+) intake causes a rapid dephosphorylation, and hence inactivation, of the thiazide‐sensitive NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT). Based on experiments in heterologous expression systems, it was proposed that changes in extracellular K+ concentration ([K+]ex) modulate NCC phosphorylation via a Cl−‐dependent modulation of the with no lysine (K) kinases (WNK)‐STE20/SPS‐1‐44 related proline‐alanine‐rich protein kinase (SPAK)/oxidative stress‐related kinase (OSR1) kinase pathway. We used the isolated perfused mouse kidney technique and ex vivo preparations of mouse kidney slices to test the physiological relevance of this model on native DCT. We demonstrate that NCC phosphorylation inversely correlates with [K+]ex, with the most prominent effects occurring around physiological plasma [K+]. Cellular Cl− conductances and the kinases SPAK/OSR1 are involved in the phosphorylation of NCC under low [K+]ex. However, NCC dephosphorylation triggered by high [K+]ex is neither blocked by removing extracellular Cl−, nor by the Cl− channel blocker 4,4′‐diisothiocyano‐2,2′‐stilbenedisulphonic acid. The response to [K+]ex on a low extracellular chloride concentration is also independent of significant changes in SPAK/OSR1 phosphorylation. Thus, in the native DCT, [K+]ex directly and rapidly controls NCC phosphorylation by Cl−‐dependent and independent pathways that involve the kinases SPAK/OSR1 and a yet unidentified additional signalling mechanism.

Key points
In humans, excitation of peripheral chemoreceptors with systemic hypoxia causes hyperventilation, hypertension and tachycardia. However, the contribution of particular chemosensory areas (carotid vs. aortic bodies) to this response is unclear.
We showed that selective stimulation of the carotid body by the injection of adenosine into the carotid artery causes a dose‐dependent increase in minute ventilation and blood pressure with a concomitant decrease in heart rate in conscious humans. The ventilatory response was abolished and the haemodynamic response was diminished following carotid body ablation.
We found that the magnitude of adenosine evoked responses in minute ventilation and blood pressure was analogous to the responses evoked by hypoxia. By contrast, opposing heart rate responses were evoked by adenosine (bradycardia) vs. hypoxia (tachycardia).
Intra‐carotid adenosine administration may provide a novel method for perioperative assessment of the effectiveness of carotid body ablation, which has been recently proposed as a treatment strategy for sympathetically‐mediated diseases.
Abstract
Stimulation of peripheral chemoreceptors by acute hypoxia causes an increase in minute ventilation (VI), heart rate (HR) and arterial blood pressure (BP). However, the contribution of particular chemosensory areas, such as carotid (CB) vs. aortic bodies, to this response in humans remains unknown. We performed a blinded, randomized and placebo‐controlled study in 11 conscious patients (nine men, two women) undergoing common carotid artery angiography. Doses of adenosine ranging from 4 to 512 μg or placebo solution of a matching volume were administered in randomized order via a diagnostic catheter located in a common carotid artery. Separately, ventilatory and haemodynamic responses to systemic hypoxia were also assessed. Direct excitation of a CB with intra‐arterial adenosine increased VI, systolic BP, mean BP and decreased HR. No responses in these variables were seen after injections of placebo. The magnitude of the ventilatory and haemodynamic responses depended on both the dose of adenosine used and on the level of chemosensitivity as determined by the ventilatory response to hypoxia. Percutaneous radiofrequency ablation of the CB abolished the adenosine evoked respiratory response and partially depressed the cardiovascular response in one participant. The results of the present study confirm the excitatory role of purines in CB physiology in humans and suggest that adenosine may be used for selective stimulation and assessment of CB activity. The trial is registered at ClinicalTrials.gov NCT01939912.

Key points
The complexity of computational models is increasing, supported by research in modelling tools and frameworks. But relatively little thought has gone into design principles for complex models.
We propose a set of design principles for complex model construction with the Physiome standard modelling protocol CellML.
By following the principles, models are generated that are extensible and are themselves suitable for reuse in larger models of increasing complexity.
We illustrate these principles with examples including an architectural prototype linking, for the first time, electrophysiology, thermodynamically compliant metabolism, signal transduction, gene regulation and synthetic biology.
The design principles complement other Physiome research projects, facilitating the application of virtual experiment protocols and model analysis techniques to assist the modelling community in creating libraries of composable, characterised and simulatable quantitative descriptions of physiology.
Abstract
The ability to produce and customise complex computational models has great potential to have a positive impact on human health. As the field develops towards whole‐cell models and linking such models in multi‐scale frameworks to encompass tissue, organ, or organism levels, reuse of previous modelling efforts will become increasingly necessary. Any modelling group wishing to reuse existing computational models as modules for their own work faces many challenges in the context of construction, storage, retrieval, documentation and analysis of such modules. Physiome standards, frameworks and tools seek to address several of these challenges, especially for models expressed in the modular protocol CellML. Aside from providing a general ability to produce modules, there has been relatively little research work on architectural principles of CellML models that will enable reuse at larger scales. To complement and support the existing tools and frameworks, we develop a set of principles to address this consideration. The principles are illustrated with examples that couple electrophysiology, signalling, metabolism, gene regulation and synthetic biology, together forming an architectural prototype for whole‐cell modelling (including human intervention) in CellML. Such models illustrate how testable units of quantitative biophysical simulation can be constructed. Finally, future relationships between modular models so constructed and Physiome frameworks and tools are discussed, with particular reference to how such frameworks and tools can in turn be extended to complement and gain more benefit from the results of applying the principles.

Key points
Fish oil (FO), rich in omega‐3 polyunsaturated fatty acids, has beneficial effects on changes induced by obesity and partially prevents associated comorbidities.
The effects of FO on adipocytes from different adipose tissue depots in high‐fat (HF) diet induced obese mice have not been uninvestigated.
This is the first study to examine the effects of FO on changes in metabolism and adipokine production in adipocytes from s.c. (inguinal; ING) or visceral (retroperitoneal; RP) white adipose depots in a HF diet‐induced obese mice.
Unlike most studies performed previously, FO supplementation was initiated 4 weeks before the induction of obesity.
HF diet caused marked changes in ING (glucose uptake and secretion of adiponectin, tumour necrosis factor‐α and interleukin‐6 in ING) and RP (lipolysis, de novo lipogenesis and secretion of pro‐inflammatory cytokines) adipose depots.
Previous and concomitant FO administration prevented the changes in ING and RP adipocytes induced by the HF diet.
Abstract
In the present study, we investigated the effect of fish oil (FO) on metabolism and adipokine production by adipocytes from s.c. (inguinal; ING) and visceral (retroperitoneal; RP) white adipose depots in high‐fat (HF) diet‐induced obese mice. Mice were divided into CO (control diet), CO+FO, HF and HF+FO groups. The HF group presented higher body weight, glucose intolerance, insulin resistance, higher plasma total and low‐density lipoprotein cholesterol levels, and greater weights of ING and RP adipose depots accompanied by hypertrophy of the adipocytes. FO exerted anti‐obesogenic effects associated with beneficial effects on dyslipidaemia and insulin resistance in mice fed a HF diet (HF+FO group). HF raised RP adipocyte lipolysis and the production of pro‐inflammatory cytokines and reduced de novo synthesis of fatty acids, whereas, in ING adipocytes, it decreased glucose uptake and adiponectin secretion but did not change lipolysis. Therefore, the adipose depots play different roles in HF diet‐induced insulin resistance according to their location in the body. Concerning cytokine secretion, adipocytes per se in addition to white adopise tissue infiltrated leukocytes have to be considered in the aetiology of the comorbidities associated with obesity. Evidence is presented showing that previous and concomitant administration of FO can prevent changes in metabolism and the secretion of hormones and cytokines in ING and RP adipocytes induced by HF.

Key points
Unlike astrocytes in the brain, the potential role of enteric glial cells (EGCs) in the formation of the enteric neuronal circuit is currently unknown.
To examine the role of EGCs in the formation of the neuronal network, we developed a novel neuron‐enriched culture model from embryonic rat intestine grown in indirect coculture with EGCs.
We found that EGCs shape axonal complexity and synapse density in enteric neurons, through purinergic‐ and glial cell line‐derived neurotrophic factor‐dependent pathways.
Using a novel and valuable culture model to study enteric neuron–glia interactions, our study identified EGCs as a key cellular actor regulating neuronal network maturation.
Abstract
In the nervous system, the formation of neuronal circuitry results from a complex and coordinated action of intrinsic and extrinsic factors. In the CNS, extrinsic mediators derived from astrocytes have been shown to play a key role in neuronal maturation, including dendritic shaping, axon guidance and synaptogenesis. In the enteric nervous system (ENS), the potential role of enteric glial cells (EGCs) in the maturation of developing enteric neuronal circuit is currently unknown. A major obstacle in addressing this question is the difficulty in obtaining a valuable experimental model in which enteric neurons could be isolated and maintained without EGCs. We adapted a cell culture method previously developed for CNS neurons to establish a neuron‐enriched primary culture from embryonic rat intestine which was cultured in indirect coculture with EGCs. We demonstrated that enteric neurons grown in such conditions showed several structural, phenotypic and functional hallmarks of proper development and maturation. However, when neurons were grown without EGCs, the complexity of the axonal arbour and the density of synapses were markedly reduced, suggesting that glial‐derived factors contribute strongly to the formation of the neuronal circuitry. We found that these effects played by EGCs were mediated in part through purinergic P2Y1 receptor‐ and glial cell line‐derived neurotrophic factor‐dependent pathways. Using a novel and valuable culture model to study enteric neuron–glia interactions, our study identified EGCs as a key cellular actor required for neuronal network maturation.

Key points
Our understanding of the mechanisms underlying the role of hypoxia in the initiation and progression of renal disease remains rudimentary.
We have developed a method that allows wireless measurement of renal tissue oxygen tension in unrestrained rats.
This method provides stable and continuous measurements of cortical tissue oxygen tension (PO2) for more than 2 weeks and can reproducibly detect acute changes in cortical oxygenation.
Exogenous angiotensin‐II reduced renal cortical tissue PO2 more than equi‐pressor doses of phenylephrine, probably because it reduced renal oxygen delivery more than did phenylephrine.
Activation of the endogenous renin–angiotensin system in transgenic Cyp1a1Ren2 rats reduced cortical tissue PO2; in this model renal hypoxia precedes the development of structural pathology and can be reversed acutely by an angiotensin‐II receptor type 1 antagonist.
Angiotensin‐II promotes renal hypoxia, which may in turn contribute to its pathological effects during development of chronic kidney disease.
Abstract
We hypothesised that both exogenous and endogenous angiotensin‐II (AngII) can decrease the partial pressure of oxygen (PO2) in the renal cortex of unrestrained rats, which might in turn contribute to the progression of chronic kidney disease. Rats were instrumented with telemeters equipped with a carbon paste electrode for continuous measurement of renal cortical tissue PO2. The method reproducibly detected acute changes in cortical oxygenation induced by systemic hyperoxia and hypoxia. In conscious rats, renal cortical PO2 was dose‐dependently reduced by intravenous AngII. Reductions in PO2 were significantly greater than those induced by equi‐pressor doses of phenylephrine. In anaesthetised rats, renal oxygen consumption was not affected, and filtration fraction was increased only in the AngII infused animals. Oxygen delivery decreased by 50% after infusion of AngII and renal blood flow (RBF) fell by 3.3 ml min−1. Equi‐pressor infusion of phenylephrine did not significantly reduce RBF or renal oxygen delivery. Activation of the endogenous renin–angiotensin system in Cyp1a1Ren2 transgenic rats reduced cortical tissue PO2. This could be reversed within minutes by pharmacological angiotensin‐II receptor type 1 (AT1R) blockade. Thus AngII is an important modulator of renal cortical oxygenation via AT1 receptors. AngII had a greater influence on cortical oxygenation than did phenylephrine. This phenomenon appears to be attributable to the profound impact of AngII on renal oxygen delivery. We conclude that the ability of AngII to promote renal cortical hypoxia may contribute to its influence on initiation and progression of chronic kidney disease.

Key points
The cortical collecting duct (CCD) plays an essential role in sodium homeostasis by fine‐tuning the amount of sodium that is excreted in the urine.
Ex vivo, the microperfused CCD reabsorbs sodium in the absence of lumen‐to‐bath concentration gradients.
In the present study, we show that, in the presence of physiological lumen‐to‐bath concentration gradients, and in the absence of endocrine, paracrine and neural regulation, the mouse CCD secretes sodium, which represents a paradigm shift.
This secretion occurs via the paracellular route, as well as a transcellular pathway that is energized by apical H+/K+‐ATPase type 2 pumps operating as Na+/K+ exchangers.
The newly identified transcellular secretory pathway represents a physiological target for the regulation of sodium handling and for anti‐hypertensive therapeutic agents.
Abstract
In vitro microperfusion experiments have demonstrated that cortical collecting ducts (CCDs) reabsorb sodium via principal and type B intercalated cells under sodium‐depleted conditions and thereby contribute to sodium and blood pressure homeostasis. However, these experiments were performed in the absence of the transepithelial ion concentrati